A carassius auratus cell line and its application

Abstract

The present application relates to a kind of goldfish milt cell line and its application, belong to aquatic animal cell technical field.The preservation number of the present application goldfish milt cell line RcGFSB is: GDMCC NO:68000.The present application first successfully establishes the stable cell line from goldfish milt tissue, the cell line has good proliferation ability and is stable, can be long-term subculture and freeze storage;The goldfish milt cell line is sensitive to a variety of aquatic animal viruses, especially to the most serious virus pathogen of carp herpesvirus II type currently harmful to crucian carp and its variety, provides key experimental material for the isolation culture of the specificity virus and other aquatic pathogens of crucian carp and its variety, pathogenesis research and antiviral drug screening;The cell line has good exogenous gene expression capacity, can be used as fish bioreactor for recombinant protein production and gene engineering vaccine research and development, has important application prospect.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal cell technology, and in particular to a swim bladder cell line of the Ranchu goldfish and its applications. Background Technology

[0002] Ranchu goldfish ( Carassius auratus auratus ) is a precious ornamental goldfish strain developed through long-term artificial breeding, and is a type of crucian carp ( Carassius auratus A variant of , belonging to the order Cypriniformes, family Cyprinidae, genus Carapax ( Carassius Its core morphological characteristics include a smooth back without a dorsal fin, highly developed head sarcoma, a significantly arched back, a short and stocky body, and a short and spread tail fin. Overall, it has unique aesthetic and genetic research value and is one of the important model organisms for fish morphogenesis and genetic breeding research.

[0003] Cell culture is a core technology in life sciences, cell engineering, genetic engineering, and biomedical engineering. The construction of cell lines provides crucial foundational materials for numerous research fields, including virology, vaccinology, genetics, and environmental toxicology. Particularly in fish virology research, the isolation and identification of virus strains using sensitive cell lines is the gold standard recommended by the World Organisation for Animal Health (WOAH), providing an indispensable experimental platform for the etiology, immunology, and vaccine development of viral diseases in fish.

[0004] Although more than ten types of goldfish cell lines have been reported both domestically and internationally, only the pelvic fin cell line of the common goldfish (GFF), the brain cell line of the goldfish (GFB), the caudal fin cell line of the gilled goldfish (RyuF-2), the gill cell line of the fantail goldfish (FtGG), the brain cell line of the fantail goldfish (FtGB), and the liver cell line of the fantail goldfish (FtGL) have the ability to stably culture carp herpesvirus type II. Cyvirus cyprinidallo2 There have been no reports on CyHV-2, a virus that has the greatest impact on the crucian carp industry, including goldfish, which limits in-depth virological and vaccinological research on CyHV-2. To date, there have been no reports on the swim bladder cell line of Ranchu goldfish. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Ranchu goldfish swim bladder cell line and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a Ranchu goldfish ( Carassius auratus auratusThe swim bladder cell line RcGFSB, with accession number GDMCC NO: 68000, was deposited on March 25, 2026 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] This invention obtains the swim bladder cell line RcGFSB from the swim bladder organ tissue of Ranchu goldfish. Its karyotype conforms to the somatic cell karyotype of goldfish (2n=4x=100) and it is sensitive to various fish viruses. This cell line grows well, can be stably passaged for more than 130 generations, has a passage doubling time of 48-72 hours, and its growth characteristics do not change significantly after cryopreservation and thawing.

[0008] Secondly, the present invention provides the application of the RcGFSB swim bladder cell line of the Ranchu goldfish as a host cell for the study of fish viruses.

[0009] Viral infection experiments have demonstrated that the RcGFSB swim bladder cell line of Ranchu goldfish, when inoculated with carp herpesvirus type II (… Cyvirus cyprinidallo2 CyHV-2), Cyril viremia virus (CyHV-2), Cyril viremia virus (CyHV-2) Sprivivirus cyprinus SVCV), mandarin frog virus ( Ranavirus micropterus1 After six passages, the RcGFSB cell line consistently exhibited significant cytopathic effects after 72 hours. Therefore, the RcGFSB swim bladder cell line of the Ranchu goldfish in this invention is sensitive to these viruses and can be used as a host cell for studying fish viruses.

[0010] Thirdly, the present invention provides the application of the described Ranchu goldfish swim bladder cell line in the isolation and / or culture of fish viruses.

[0011] Fourthly, the present invention provides the application of the described Ranchu goldfish swim bladder cell line in the detection of fish viruses for non-disease diagnostic purposes.

[0012] Fifthly, the present invention provides the application of the described Ranchu goldfish swim bladder cell line as an in vitro virus infection model for aquatic animals.

[0013] The RcGFSB swim bladder cell line of Ranchu goldfish was inoculated with carp herpesvirus type II, carp spring viremia virus, and mandarin frog virus. After six passages, the RcGFSB cells consistently showed significant cytopathic effects after 72 hours. Therefore, the RcGFSB swim bladder cell line of Ranchu goldfish in this invention is sensitive to these viruses and can be used for the isolation, culture, detection of viruses in fish for non-disease diagnostic purposes, and as an in vitro viral infection model for aquatic animals.

[0014] Sixthly, the present invention provides the application of the described Ranchu goldfish swim bladder cell line in expressing exogenous genes.

[0015] In this invention, the swim bladder cell line RcGFSB of Ranchu goldfish was transfected with the pEGFP-N3 plasmid expressing the exogenous gene EGFP and the recombinant virus CyHV-2-∆57-CP, which is a fusion protein of EGFP and NNV-CP. The results showed that the cells could express large amounts of EGFP and the EGFP-NNV-CP fusion protein. Therefore, the cells of this invention can be used to express exogenous genes.

[0016] Furthermore, the vector expressing the exogenous gene is a plasmid vector or a viral vector.

[0017] In a seventh aspect, the present invention provides a method for preparing the RcGFSB swim bladder cell line of the Ranchu goldfish, comprising the following steps: Tissue blocks of the swim bladder organ of Ranchu goldfish were cultured in DMEM medium until cells migrated out. The culture medium was then replaced with fresh medium until the cell confluence reached more than 90% to obtain primary cells. The culture medium in the primary cells was discarded, and the primary cells were digested with enzymes to obtain single cells. After obtaining single cells, culture medium was added and the cells were passaged to obtain the Ranchu goldfish swim bladder cell line RcGFSB.

[0018] Eighthly, the present invention provides a method for preserving the RcGFSB swim bladder cell line of Ranchu goldfish, comprising the following steps: taking a suspension of RcGFSB cells in the logarithmic growth phase of the Ranchu goldfish swim bladder cell line, centrifuging, resuspending the cell pellet in a cryopreservation solution pre-cooled at 4-5℃, transferring the cell suspension to -70~-90℃ and freezing overnight, and transferring it to liquid nitrogen for long-term preservation the next day; the cryopreservation solution consists of 90% fetal bovine serum and 10% dimethyl sulfoxide by mass percentage.

[0019] Ninthly, the present invention provides a method for culturing the RcGFSB swim bladder cell line of Ranchu goldfish, comprising the following steps: inoculating the RcGFSB swim bladder cell line of Ranchu goldfish into a culture medium and culturing it at 25-30°C; wherein the culture medium is DMEM medium containing 1500-2500 IU / mL penicillin, 1-5 μg / mL streptomycin, 2-8 μg / mL amphotericin B and 15%-25% fetal bovine serum.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention has for the first time successfully established a stable cell line derived from the swim bladder tissue of Ranchu goldfish. This cell line has good and stable proliferation capacity and can be passaged and cryopreserved for a long time, filling the gap in swim bladder tissue cell resources of this species.

[0021] 2. The Ranchu goldfish swim bladder cell line provided by this invention is sensitive to a variety of aquatic animal viruses, especially to carp herpesvirus type II, which is currently the most serious viral pathogen affecting crucian carp and its variants (such as goldfish). It provides key experimental materials for the isolation and culture of specific viruses and other aquatic pathogens, including but not limited to crucian carp and its variants (such as goldfish), the study of pathogenic mechanisms, and the screening of antiviral drugs.

[0022] 3. The cell line provided by this invention has good exogenous gene expression ability and can be used as a fish bioreactor for recombinant protein production and genetic engineering vaccine research and development, which has important application prospects. Attached Figure Description

[0023] Figure 1 Figure 1 shows the microscopic observation results of the RcGFSB swim bladder cell line of Ranchu goldfish at different stages (Figure A is the primary swim bladder cells of Ranchu goldfish; Figure B is the swim bladder cells of the 3rd generation Ranchu goldfish; Figure C is the swim bladder cells of the 40th generation Ranchu goldfish; Figure D is the swim bladder cells of the 110th generation Ranchu goldfish; scale bar = 100 μm).

[0024] Figure 2 A schematic diagram illustrating the effect of different bovine fetal serum (FBS) concentrations (5%, 10%, and 15%) on the growth of the RcGFSB swim bladder cell line of Ranchu goldfish; Figure 3 This is a schematic diagram of the karyotype analysis of the 40th generation of the RcGFSB swim bladder cell line of Ranchu goldfish (Figure A shows chromosome composition; Figure B shows chromosome number distribution). Figure 4 Schematic diagram of cytopathic effect (CPE) of RcGFSB swim bladder cell line in Ranchu goldfish after 72 h of infection with different viruses (Figure A shows RcGFSB inoculated with CyHV-2; Figure B shows RcGFSB inoculated with SVCV; Figure C shows RcGFSB inoculated with MRV; Figure D shows the negative control; scale bar = 100 μm). Figure 5 Transmission electron microscopy (TEM) images of RcGFSB swim bladder cell line from Ranchu goldfish infected with different viruses 72 h later (Figures A, B, and C show the results of RcGFSB inoculated with CyHV-2, SVCV, and MRV at 15,000× magnification, scale bar = 1 μm). Figure 6 Fluorescence microscopy results of RcGFSB swim bladder cell line transfected with pEGFP-N3 plasmid 24 h later (scale bar = 100 μm). Figure 7 Fluorescence microscopy results of different stages of infection with the recombinant virus CyHV-2-∆57-CP, which is a chimeric EGFP and NNV-CP fusion protein gene, in the swim bladder cell line RcGFSB of Ranchu goldfish. Detailed Implementation

[0025] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0026] Table 1. Comparison of English Abbreviations and Chinese Names Example 1: Construction and preservation of the RcGFSB swim bladder cell line from Ranchu goldfish (1) Primary culture: Select a healthy Ranchu goldfish weighing approximately 8 g. First, rinse off surface dirt and mucus with running water. Then, anesthetize the fish by placing it in crushed ice for 10 minutes. Next, disinfect it in 75% alcohol for 30 seconds. Under aseptic conditions, remove the intact swim bladder. Using sterile forceps, immerse the swim bladder in an HBSS solution containing 2000 U / mL penicillin, 2 mg / mL streptomycin, and 5 μg / mL amphotericin B (i.e., 20% triple antibiotics), gently rinsing for 3 minutes. Then, transfer the swim bladder to a fresh HBSS solution containing the triple antibiotics, repeating the rinsing process three times. Finally, use sterile scissors to cut the swim bladder into pieces approximately 1 mm in size. 3 Tissue blocks of the desired size were evenly placed in one well of a 6-well plate. After incubation at 27°C upside down for 2 hours, 3 mL of DMEM culture medium containing 20% ​​FBS and 2% triple antibodies (containing 2000 U / mL penicillin, 2 mg / mL streptomycin, and 5 μg / mL amphotericin B) was slowly added along the well wall. The plate was then incubated at 27°C in a 5% CO2 incubator. Once cell migration began, half of the culture medium was replaced every 4 days until cell confluence reached over 90%, yielding the desired cell type. Figure 1 A shows the swim bladder cells of the primary Ranchu goldfish.

[0027] (2) Subculture: Once the primary cultured cells have reached a confluence of over 90%, discard the old culture medium, wash once with HBSS solution, add 500 μL of 0.25% trypsin and let it stand for 2–3 min to digest. After the cells become round and begin to detach, add 2 mL of DMEM culture medium as described in step (1) to stop the digestion and gently pipette them into single cells. Then transfer them to T25 cell culture flasks and add culture medium to make up to 6 mL. Place them in a constant temperature incubator at 27℃ and 5% CO2 concentration for culture. In subsequent passage cultures, cells were cultured in flasks at a ratio of 1:2 or 1:3 according to their growth rate. After 5 passages, antibiotics were no longer added to the culture medium. After 20 passages, the proportion of FBS in the culture medium was reduced to 15%. After 30 passages, the proportion of FBS in the culture medium was reduced to 10%. After 40 passages, a stable RcGFSB swim bladder cell line was obtained. This line was then passaged in flasks at a 1:3 ratio, and confluence reached over 90% within 3 days, yielding the desired result. Figure 1 The cell morphology shown in C is mainly fibroblast-like swim bladder cells of the Ranchu goldfish. The morphology of the swim bladder cells of a Ranchu goldfish that has been passaged 110 times is as follows. Figure 1 As shown in D.

[0028] (3) Cell cryopreservation: When the cells have grown well and reached approximately 90% confluence, cell digestion and termination are performed using the method described in step (2). After centrifugation at 200×g for 3 min, the culture medium is discarded to collect the cells. Cell freezing solution (10% DMSO and 90% FBS) is added, and the cells are gently resuspended by pipetting. The cells are then injected at approximately 2.5×10⁻⁶ ppm. 6 A concentration of cells / mL was transferred to 1 mL into each cell cryopreservation tube, labeled, and placed in a programmed cooling box. After being placed in a -80°C freezer for more than 6 hours, the tubes were then transferred to liquid nitrogen for long-term storage.

[0029] (4) Cell resuscitation: Quickly place the cell cryopreservation tube in clean warm water at 35±2℃ and shake it to thaw it rapidly. Then slowly add the cell suspension to a centrifuge tube containing 10 mL of the culture medium described in step (1). Centrifuge at 200×g for 3 min to collect the cells. Add 5 mL of the above culture medium again and gently resuspend the cells into single cells. Then transfer them to T25 cell culture flasks and incubate them in a constant temperature incubator at 27℃ and 5% CO2 concentration. The next day, depending on the cell density, choose to replace the culture medium with fresh medium or perform cell passage.

[0030] The Ranchu goldfish obtained by this invention ( Carassius auratus auratusThe swim bladder cell line RcGFSB was deposited on March 25, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510075, China, with accession number GDMCC NO: 68000.

[0031] Example 2: Growth curve determination of the RcGFSB swim bladder cell line of Ranchu goldfish After passaged three times using DMEM culture medium containing 5% FBS, the RcGFSB swim bladder cell line of Ranchu goldfish was digested and diluted to approximately 5 × 10⁻⁶ cells using DMEM culture medium containing 5%, 10%, and 15% FBS, respectively. 4 Cell suspensions of 3 cells / mL were seeded into several 6-well plates, with 3 mL of the cell suspension added to each well. The plates were then incubated in a constant temperature incubator at 27°C and 5% CO2. Every 24 hours, 3 wells of cells were randomly selected from each serum concentration gradient group, digested with trypsin, and counted using a hemocytometer. Cell counting was performed continuously for 6 days, and cell growth curves were plotted at different serum concentration gradients.

[0032] The results are as follows Figure 2 As shown, the growth rate of Ranchu goldfish swim bladder cell lines cultured in DMEM medium containing 10% and 15% FBS was not significantly different, while the growth rate was slightly slower under the condition of 5% FBS.

[0033] Example 3 Karyotype analysis of the RcGFSB swim bladder cell line from Ranchu goldfish When the RcGFSB swim bladder cell line was passaged to the 40th generation, colchicine was added to a final concentration of 0.2 µg / mL and incubated for 5–6 h. Cells were collected using the method described in step (3) of Example 1. 8 mL of 0.075 M potassium chloride solution preheated to 37°C was added, and the cells were hypotonic at 37°C for 40 min. Cells were collected by centrifugation at 500×g for 5 min, and 2 mL of freshly prepared Carnoy's fixative (methanol:acetic acid volume ratio = 3:1) was added and gently mixed for pre-fixation for 5 min. Cells were collected by centrifugation at 500×g for 10 min, and the residue was aspirated to 0.5 mL of fixative. Another 5 mL of fixative was added and gently mixed, and the cells were incubated at room temperature in the dark for 40 min. This fixation step was repeated once. Cells were collected by centrifugation at 500×g for 10 min, and the residue was aspirated to 0.5 mL of fixative. The cells were gently resuspended by pipetting. The cell suspension was gradually added dropwise from a height of 40–60 cm onto a pre-cooled (-20°C) glass slide tilted at approximately 45°. After the fixative had evaporated, freshly prepared 10% Giemsa staining solution was added and stained for 20–30 min. The staining solution was slowly rinsed with tap water, and the slide was air-dried at room temperature. The chromosome droplets were observed under a 100× oil immersion microscope, and the chromosomes in 100 metaphase stages were counted and analyzed.

[0034] The results are as follows Figure 3 As shown, Figure 3 A shows the chromosome composition of the RcGFSB swim bladder cell line from the genus *Rhododendron simsii*. Figure 3 B shows that the karyotype of the RcGFSB swim bladder cell line from Ranchu goldfish conforms to the somatic karyotype of goldfish with 2n=4x=100, accounting for 24%.

[0035] Example 4: Sensitivity test of the RcGFSB swim bladder cell line from Ranchu goldfish to different viruses. (1) CPE observation: When the confluence of RcGFSB in the epicellular cells of Ranchu goldfish reaches 50-60%, carp herpesvirus type II (CGFSB) is selected. Cyvirus cyprinidallo2 CyHV-2), Cyril viremia virus (CyHV-2), Cyril viremia virus (CyHV-2) Sprivivirus cyprinus SVCV) and mandarin frog virus ( Ranavirus micropterus1 Cells were seeded with MRV (MOI=0.1) and incubated at 25°C for 2 h for adsorption. The old culture medium was then discarded and replaced with DMEM maintenance medium containing 2% FBS. The cells were then cultured in a constant temperature incubator at 25°C and 5% CO2. When the cytopathic effect reached 80–90%, the virus solution was harvested after one freeze-thaw cycle and passaged 6 times in the RcGFSB cell line as described above.

[0036] The results are as follows Figure 4 After six passages in RcGFSB cells, the aforementioned viruses consistently exhibited a significant cytopathic effect (CPE) after 72 hours: CyHV-2-induced CPE resulted in most cells shrinking, rounding, and detaching, while a small number of cells showed swelling, fusion, and vacuolization. Figure 4 A); and SVCV ( Figure 4 B) and MRV ( Figure 4 C) The resulting CPE is similar, mainly characterized by cell shrinkage, rounding, and shedding.

[0037] (2) Transmission electron microscopy observation: Take the cells infected with each strain of virus (6th generation) in step (1) above, discard the culture medium, rinse with HBSS and discard it, scrape and collect the cells with a cell scraper, and prepare ultrathin cell sections for transmission electron microscopy observation.

[0038] The results are as follows Figure 5 A and Figure 5 As shown in D, in RcGFSB cells infected with CyHV-2, enlarged nuclei contain numerous crystalline arrays of nucleocapsids and empty capsids (white arrows), and mature viral particles with envelopes are visible in the cytoplasm (red arrows); Figure 5 B and Figure 5As shown in Figure E, inclusion bodies containing a large number of viral particles and budding viral particles are visible in RcGFSB cells infected with SVCV; Figure 5 C and Figure 5 As shown in Figure F, in RcGFSB cells infected with MRV, "virus factories" containing a large number of virus particles and budding virus particles can be seen.

[0039] The above results indicate that the RcGFSB swim bladder cell line of Ranchu goldfish is sensitive to CyHV-2, SVCV, and MRV viruses, and can be used as a tool for detecting, isolating, culturing, and studying these viruses.

[0040] Example 5: Test of exogenous gene expression in the RcGFSB swim bladder cell line of Ranchu goldfish (1) Plasmid vector: RcGFSB swim bladder cells of Ranchu goldfish with good growth were seeded into 6-well plates. When the cell confluence reached 70%, cells were transfected using FuGENE® HD transfection reagent and pEGFP-N3: ​​1 μg of endotoxin-free pEGFP-N3 plasmid was added to 100 μL of Opti-MEM and mixed thoroughly. Then, 3 μL of transfection reagent was added and mixed thoroughly. The mixture was incubated at room temperature for 15 min. The mixture was then dropped into the cells of the 6-well plate, gently shaken to mix, and incubated in a constant temperature incubator at 27°C and 5% CO2 concentration for 4 h. The medium was then replaced with fresh DMEM containing 10% FBS.

[0041] The results are as follows Figure 6 As shown, a large number of cells expressing EGFP were observed 24 h after transfection, indicating that the RcGFSB swim bladder cell line of Ranchu goldfish can be used for the expression of exogenous genes based on plasmid vectors.

[0042] (2) Viral vector: The recombinant virus CyHV-2-∆57-CP (CyHV-2 with EGFP and NNV-CP fusion protein genes) constructed and preserved in our laboratory was used to inoculate the RcGFSB swim bladder cell line of Ranchu goldfish according to the method described in Example 4. The cells were observed and photographed daily.

[0043] The results are as follows Figure 7 As shown, since NNV-CP has a nuclear localization sequence, green fluorescence can be observed in the cell nucleus 48 h after inoculation with recombinant virus. As time increases, the green fluorescence intensifies and spreads to the entire cell. After 120 h of inoculation with recombinant virus, green fluorescence can be observed in more than 90% of the cells, indicating that the RcGFSB swim bladder cell line of Ranchu goldfish can be used for the expression of exogenous genes based on viral vectors.

[0044] The above results indicate that the RcGFSB swim bladder cell line of Ranchu goldfish can express exogenous genes in multiple ways, and can be applied to gene function research, recombinant virus construction, and recombinant protein expression.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A swim bladder cell line RcGFSB from Ranchu goldfish, characterized in that, Its accession number is GDMCC NO: 68000. It was deposited on March 25, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of the Ranchu goldfish swim bladder cell line of claim 1 as a host cell for studying fish viruses.

3. The application of the Ranchu goldfish swim bladder cell line of claim 1 in the isolation and / or culture of fish viruses.

4. The application of the Ranchu goldfish swim bladder cell line according to claim 1 in the detection of fish viruses for non-disease diagnostic purposes.

5. The application of the Ranchu goldfish swim bladder cell line according to claim 1 as an in vitro viral infection model for aquatic animals.

6. The application of the Ranchu goldfish swim bladder cell line according to claim 1 in expressing exogenous genes.

7. The application of claim 6, characterized in that, The vector expressing the exogenous gene is a plasmid vector or a viral vector.

8. The method for preparing the Ranchu goldfish swim bladder cell line according to claim 1, characterized in that, Includes the following steps: Tissue blocks of the swim bladder organ of Ranchu goldfish were cultured in DMEM medium until cells migrated out. The culture medium was then replaced with fresh medium until the cell confluence reached more than 90% to obtain primary cells. The culture medium in the primary cells was discarded, and the primary cells were digested with enzymes to obtain single cells. After obtaining single cells, culture medium was added and the cells were passaged to obtain the Ranchu goldfish swim bladder cell line.

9. The method for preserving the swim bladder cell line of Ranchu goldfish as described in claim 1, characterized in that, Includes the following steps: Take the cell suspension of the Ranchu goldfish swim bladder cell line in the logarithmic growth phase, centrifuge, resuspend the cell pellet in cryopreservation solution pre-cooled at 4-5℃, transfer the cell suspension to -70~-90℃ and freeze overnight, then transfer it to liquid nitrogen for long-term storage the next day; the cryopreservation solution is composed of 90% fetal bovine serum and 10% dimethyl sulfoxide by mass percentage.

10. The method for culturing the Ranchu goldfish swim bladder cell line according to claim 1, characterized in that, Includes the following steps: The swim bladder cell line of Ranchu goldfish was inoculated into a culture medium and cultured at 25-30℃. The culture medium was DMEM medium containing 1500-2500 IU / mL penicillin, 1-5 μg / mL streptomycin, 2-8 μg / mL amphotericin B and 15%-25% fetal bovine serum.

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