A yellowfin seabream kidney cell line and its application

Abstract

The present invention discloses a yellowfin seabream kidney cell line and its application. The deposit number of the yellowfin seabream kidney cell (yellowfin seabream Kindey cell line) YSK is: GDMCC No: 65080. The yellowfin seabream kidney cell line YSK is constructed through primary culture and subculture, and its cell morphology is mainly fibroblast-like, the cells grow rapidly, and have been stably passaged for more than 60 generations. It has good vitality after ultra-low temperature preservation. The yellowfin seabream kidney cell line YSK cell line can be used for the isolation of marine fish viruses, the pathogenic mechanism of viral infection and the study of exogenous gene functions. It also provides a good in vitro cell amplification system for the preparation of yellowfin seabream dephosphorylation disease virus SDDV vaccine.

Description

Technical Field

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

[0002] The yellowfin sea bream (Acanthopagrus latus), commonly known as yellow-legged croaker, red-winged croaker, or yellow croaker, is a species of fish in the genus Acanthopagrus of the family Sparidae in the order Perciformes. It is an important commercial fish species along the coast of South China. As a shallow, warm-water, demersal fish, yellowfin sea bream is adaptable to changes in salinity and can thrive in waters of varying salinity. Its nutritious nature, delicious meat, and high economic value make it a popular edible fish. However, with the development of yellowfin sea bream aquaculture, frequent disease outbreaks have posed challenges and obstacles to the industry's development. Viral diseases are a major infectious disease threatening yellowfin sea bream aquaculture. Scaledrop syndrome virus (SDDV), a major pathogen causing ascites in yellowfin sea bream, belongs to the genus Cytosomavirus of the family Iridoviridae. Furthermore, SDDV can infect both juvenile and adult Asian sea bass, resulting in a mortality rate of approximately 40-50%. Currently, there are no effective treatments or control measures for isolates of SDDV.

[0003] Fish cell lines, as ideal in vitro culture systems, have been widely used in basic and applied research, including virus isolation and identification, viral pathogenic mechanisms, host gene function, and large-scale viral vaccine preparation, due to their numerous advantages, including low cost, ease of operation, good reproducibility, and controllable conditions. With the interdisciplinary integration of disciplines, cell lines are also being used in research fields such as immunology, toxicology, genetic developmental biology, and oncology. Compared with other marine fish, relatively few cell lines have been established for yellowfin sea bream, and no yellowfin sea bream kidney cell lines have been reported. This significantly limits in vitro studies of the pathogenic mechanisms of yellowfin sea bream virus infection and the development of inactivated viral vaccines. Therefore, it is necessary to establish yellowfin sea bream cell lines that are sensitive to different viruses to provide excellent experimental materials for studying viral infection mechanisms, viral gene function, and vaccine prevention and control.

[0004] This invention is supported by the project title "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 exogenous genes.

[0008] The third object of the present invention is to provide the use of the yellowfin sea bream kidney cell line as a host cell for studying marine fish viruses, wherein the virus is yellowfin sea bream dephosphovirus, Singapore grouper iridovirus or neural necrosis 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 and / or detection of marine fish viruses for non-disease diagnosis purposes, wherein the virus is yellowfin sea bream dephosphovirus, Singapore grouper iridovirus or neural necrosis virus.

[0010] The fifth object of the present invention is to provide the use of the above-mentioned yellowfin seabream kidney cell line in the isolation of marine fish viruses, wherein the virus is yellowfin seabream dephosphovirus, Singapore grouper iridovirus or neural necrosis 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 dephosphovirus, 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 dephosphating disease virus.

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

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

[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 obtained the yellowfin sea bream kidney cell line YSK, whose cell morphology is mainly fibroblast-like, the cells grow rapidly, have been stably passaged for more than 60 generations, and have good vitality after ultra-low temperature storage, laying the foundation for the preservation of yellowfin sea bream genetic germplasm resources.

[0018] 2. The method for constructing a yellowfin sea bream kidney cell line provided by the present invention uses a digestion and culture method for primary cell culture. No other growth factors are required during the culture of primary and passaged cells, and the culture conditions are not stringent.

[0019] 3. The yellowfin sea bream kidney cell line provided by the present invention is sensitive to 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 Provincial Microbial Culture Collection Center (GDMCC) on August 30, 2024. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, with the deposit number: 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 morphologies of the 20th and 60th passages of YSK cells, respectively.

[0024] Figure 2 Graph showing the growth curves of YSK cells under different culture conditions in Example 2 of the present invention; A represents the effect of different temperatures on cell growth; and 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; 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 3. 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, objectives, 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 embodiments described are only part of the embodiments of the present invention, not all of them. It should be pointed out that based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this 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 cultured yellowfin sea bream (Acanthopagrus latus) with a body length of 5 cm.

[0031] (2) 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 manufacturer's instructions. 0.266% NaCl and 5 mM HEPES were added, and the pH was adjusted to 7.4. After filtering through a 0.22 μm filter, the medium was aliquoted and stored at 4°C until use. For later use, different concentrations of fetal bovine serum or a double antibiotic (penicillin-streptomycin) were added as needed.

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

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

[0035] Passage cell culture medium: The culture medium used for cells of passages 1-10 is the primary cell culture medium; after the 10th cell passage, the serum concentration of the passage 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 juvenile fish. 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 suspension containing about 4×10 6 The cell suspension was transferred into a 25 cm 2 Place the cells in a culture flask. Add 5 mL of primary cell culture medium to each flask. Place the flask in a 28°C incubator. Replace the culture medium by half every four days. Observe and record cell adhesion and growth using a phase contrast microscope. On the second day of primary culture, primary cells begin to adhere to the flask. Initially, the cells exhibit diverse morphologies, including both fibrous and epithelial cells. After 10 days, the primary cells cover approximately 85% of the flask bottom, and subculture begins.

[0038] (5) Subculture

[0039] During the initial passage, transfer the culture medium in the primary culture cell bottle to a sterile culture bottle. Use 1mL0.25% trypsin-EDTA digestion solution to digest the monolayer cells at room temperature, mix 5mL of fresh passage cell culture medium and 5mL of primary cell culture medium and add them to the culture bottle of monolayer cells, gently tap the bottle wall to make the monolayer cells fall off into the culture medium, and then gently blow with 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 passage, passage is performed once every 5 days according to the above method, and the culture medium is primary cell culture medium. When it is passed to the 10th generation, the fetal bovine serum in the passage cell culture medium is reduced to 10%, and the antibiotic concentration is 100IU / mL penicillin and 100μg / mL streptomycin; passage is performed once every 3 days. The primary cells of the yellowfin sea bream kidney constructed in this example are fibroblast-like and epithelial-like. As the number of passages increases, the cell morphology becomes spindle-shaped, and fibroblasts are the main cells ( Figure 1 The cells are now morphologically stable and have been continuously passaged for more than 60 generations. They are named the yellowfin seabream kidney cell line (YSK).

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

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

[0042] After one month of cryopreservation, the frozen cells were revived. The cryovials were removed from liquid nitrogen and quickly thawed in a 37°C water bath. The cell suspension was centrifuged at 1000 rpm for 10 minutes to collect the cell pellet, which was then resuspended in 1 mL of cell culture medium. A small amount of the cell suspension was stained with 0.4% trypan blue for 5 minutes, and the number of live and dead cells was counted using a hemocytometer. The remaining cell suspension was mixed with 4 mL of cell culture medium and transferred to a culture flask for incubation at 28°C. Cell adhesion and growth were observed. The cell viability of YSK cells after cryopreservation was 81%, and the morphology and proliferation of the surviving cells after attachment were similar to those before cryopreservation.

[0043] The yellowfin seabream kidney cell line YSK was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on August 30, 2024. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, with the deposit number: 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 cell growth at different culture temperatures. The specific operation was as follows: 6×10 4 Cells were seeded into 12-well plates in 1 mL of L-15 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 removed from each experimental group on days 1, 3, 5, and 7 of culture, and the cells were counted with a hemocytometer to plot cell growth curves. 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 4Cells were seeded into 12-well plates, and L-15 medium (1 mL) containing different concentrations of fetal bovine serum was added to each well. The concentrations of fetal bovine serum were set at 5%, 10%, and 15% by volume, respectively. The cells were 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 Figure B, cell growth rate is proportional to the added serum concentration. On day 3 of culture, medium containing 15% and 10% serum significantly promoted cell proliferation, while 10% serum maintained normal cell growth. Cell proliferation was significantly slowed at 5% serum, with the cell count on day 3 being only one-third of that in the medium containing 10% serum. For cost-effectiveness, L-15 medium containing 10% serum by volume is the preferred medium for YSK cell culture starting from passage 10.

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

[0050] YSK cells at passage 25 in Example 1 were seeded into a 24-well cell culture plate and cultured overnight (the culture medium used was the passaged cell culture medium described in Example 1). Transfection was initiated when the cells reached a confluence of more than 85% of the monolayer. Lipofectamine 2000 (Ivitrogen) was used as the transfection reagent, and pEGFP-N3 was transfected into the cells according to the reagent's instructions. 24 hours after transfection, the expression of green fluorescent protein in the cells was observed under a fluorescence microscope. 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 exogenous genes in YSK cells, and the YSK cell line can be used to study the functions of exogenous 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 passaged cell culture medium described in Example 1). The yellowfin sea bream dephosphodiesterase virus SDDV-ZH strain, neural necrosis virus RGNNV and Singapore grouper iridovirus SGIV suspensions preserved in the laboratory were added to the culture medium. 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. Typical characteristics of cells infected with SGIV and SDDV-ZH strains are rounding, increased refractive index, and individual rounded cells scattered across the cell monolayer. 36 hours after infection, the rounded cells shed, forming cavities in the cell monolayer. Vacuoles of varying sizes appeared in the cytoplasm of RGNNV-infected cells. 36 hours after infection, the cells necroticly shed, forming gaps in the cell monolayer. In contrast, uninfected cells observed at 24 hours showed no significant changes in cell morphology. This indicates that infection with different marine fish viruses can induce significant cytopathic effects in YSK cells.

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

[0055] The 35th generation YSK cells of Example 1 were infected with SDDV-ZH, RGNNV and SGIV according to the method of step 1. The cells were harvested at different time points after virus infection (SDDV: 24h, 48h and 96h; RGNNV: 12h, 24h and 36h; SGIV: 12h, 24h and 36h) and lysed in RIPA buffer (ThermoFisher). The expression of viral proteins in infected cells was detected by western blotting. The experimental procedure is as follows: infected and uninfected cell samples were denatured by boiling, separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane containing protein was stained with 5% skim milk (100mL PBS + 0.1% Tween 20 (PBST, v / v) + 5g skim milk powder (BD Biosciences). Difco) were 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, prokaryotic antigen SGIV-MCP) Expression product, Wuhan Jinkairui Company was commissioned to prepare the antibody) or anti-RGNNV-CP antibody (1:3000, antigen RGNNV-CP prokaryotic expression product, Wuhan Jinkairui Company was commissioned to prepare the antibody). 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, ECL color development solution (Bioscience) was used for color development in a Tanon 5200 automatic chemiluminescence imaging system.

[0056] The results are as follows Figure 4B, In the cell samples infected with SDDV-ZH, the expression of viral protein MCP increased significantly with time from 24h to 96h after infection. Similarly, in the cell samples infected with RGNNV and SGIV, the expression of viral capsid protein CP and major capsid protein MCP gradually increased from 12h to 36h after 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] YSK cells from passage 35 of Example 1 were infected with SDDV-ZH according to step 1 of Example 4. Cells were harvested 48 hours after viral infection and centrifuged at 2000 rpm for 10 minutes. The cell pellet was fixed with 2.5% glutaraldehyde for 1 hour at 4°C. The fixative was discarded, and the cells were rinsed three times with PBS for 5 minutes each. The cells were then fixed with 1% (v / v) osmium phosphate for 1 hour at 4°C, and dehydrated with a gradient of ethanol (50%, 70%, 80%, 90%, 100%, and 100% by volume) for 10 minutes. The cells were then embedded in Epon 812 epoxy resin. The samples were then ultrathinly sectioned and stained with 2% uranyl acetate and lead citrate for 1 hour each. The sections were then photographed under a transmission electron microscope (Talos L120C, ThermoFisher Scientific).

[0060] The results are as follows Figure 5 As shown in Figure A, multiple single-layer membrane vacuolar structures were observed in the cytoplasm of SDDV-infected cells. These vacuolar structures contained hexagonal virions approximately 120-130 nm in diameter. Additionally, scattered hexagonal virions were observed in the cytoplasm, demonstrating that SDDV can successfully propagate in YSK cells. Taken together, these results suggest that YSK cells can be used as an in vitro viral infection model to study the mechanisms of viral infection and pathogenicity in marine fish and investigate virus-host interactions.

[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 cells were infected with SDDV at 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 frozen and thawed three times at -20 °C. The virus was measured by the TCID 50) method to determine the titer of the virus. The specific method is as follows: ① The 42nd generation YSK cells were transferred into a 96-well cell culture plate and cultured in a constant temperature incubator at 28°C overnight. ② The samples collected at different infection time points were diluted in a 10-fold gradient. ③ Each well of cells was inoculated with 100 μL of the diluted virus-infected cell sample to be tested, and then placed in a constant temperature incubator at 28°C for infection. ④ On the 6th day after infection, the number of cell wells with CPE was observed and recorded, and the TCID of the virus-infected sample was calculated 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 24 h after infection, the virus titer in cells did not change significantly compared with that in cells infected at 0 h. At 48 h after infection, the virus titer in cells increased from 7.76×10 5 TCID 50 / mL increased to 1.66×10 6 TCID 50 As the infection time extended to 96 h, the virus in the infected cells increased to 1.48 × 10 8 TCID 50 The above results indicate that SDDV can be amplified in YSK cells to obtain a higher virus titer, that is, YSK cells provide 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 principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A yellowfin sea bream kidney cell line YSK, characterized by: The accession 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 according to claim 1 as a host cell for studying marine fish viruses, wherein the virus is yellowfin sea bream scale disease virus SDDV, Singapore grouper iridovirus SGIV or neural necrosis virus RGNNV.

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

5. Use of the yellowfin seabream kidney cell line according to claim 1 in the isolation of marine fish viruses, wherein the virus is yellowfin seabream scale disease virus SDDV, Singapore grouper iridovirus SGIV or neural necrosis virus RGNNV.

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 scale disease virus (SDDV), Singapore grouper iridovirus (SGIV), or neuronecrosis virus (RGNNV).

7. Use of the yellowfin sea bream kidney cell line according to claim 1 in the development of an inactivated vaccine against yellowfin sea bream scale disease virus (SDDV).

8. The use according to claim 7, characterized in that The yellowfin sea bream descaling disease virus (SDDV) inactivated vaccine uses virus liquid infected for 24 h to 96 h 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 phase was obtained and centrifuged. The cell pellet was resuspended in a freezing solution pre-cooled at 4-5°C, and the cell suspension was frozen at -70~-90°C overnight. The next day, the cell suspension was transferred to liquid nitrogen for long-term storage. The freezing solution was L-15 culture medium containing 20% ​​fetal bovine serum and 10% dimethyl sulfoxide by volume.

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 was inoculated into a culture medium and cultured at 25-30° C. The culture medium was an L-15 culture medium containing 100-400 IU / mL penicillin, 100-400 μg / mL streptomycin, and 10%-20% fetal bovine serum by volume.

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