Pomfret spleen cell line and construction method and application thereof
By constructing a spleen cell line from silver pomfret, the problem of frequent iridovirus infection in silver pomfret farming was solved. A highly sensitive cell line was provided for virus culture and detection, which promoted virus research and immune control and boosted the development of the aquaculture industry.
Patent Information
- Application Number
- CN202510893602.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-11-07
AI Technical Summary
In existing technologies, cell swelling during silver pomfret farming is frequently caused by iridovirus infection. The lack of effective cell lines for research and control leads to frequent disease outbreaks, hindering the commercialization of the aquaculture industry.
A silver pomfret spleen cell line was constructed by aseptically extracting spleen tissue, rinsing and disinfecting it with a buffer solution containing antibiotics and antifungal agents, and then gradually passaged it in a low-serum culture medium and digested it with trypsin to obtain a highly sensitive and stable cell line.
A highly sensitive silver pomfret spleen cell line was provided for iridovirus culture and immunoassay, reducing detection background, facilitating virology and gene expression research, solving the problem of virus diagnosis, and providing a foundation for the development of antiviral drugs.
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Figure CN120905140A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of fish cell engineering, in particular to a Pampus argenteus spleen cell line and a construction method and application thereof. BACKGROUND
[0002] Pampus argenteus belongs to Perciformes, Stromateidae and Pampus, and is one of the important economic fish species in China. Pampus argenteus in China is mainly divided into the Yellow Sea and Bohai Sea population and the East China Sea population, and is one of the main fishing objects at present. At present, breakthrough progress has been made in artificial breeding and breeding technology of Pampus argenteus in China, and gradually matures. However, due to short domestication time, great difference between breeding environment and wild environment and other reasons, Pampus argenteus in artificial breeding is susceptible to pathogenic microorganisms in the environment, diseases frequently occur in the breeding process, and diseases caused by Iridovirus infection of cell swelling have become a problem to be solved in Pampus argenteus breeding industry, which seriously hinders the process of commercialization of Pampus argenteus breeding. Cell culture is one of the important means of cell biology research methods, and at present, there are reports on cell culture and cell line construction of Pampus argenteus fin, muscle and liver tissues, and there is no report on construction and application of Pampus argenteus spleen cell line.
[0003] The spleen is an important central immune organ of fish, and is also a target organ of Iridovirus infection of cell swelling, based on which, construction of Pampus argenteus spleen cell line has important significance for pathogenesis and immune prevention and control of fish Iridovirus. SUMMARY
[0004] The purpose of the present application is to provide a Pampus argenteus spleen cell line for research on pathogenesis and immune prevention and control of fish Iridovirus.
[0005] To achieve the above purpose, the first aspect of the present application provides a Pampus argenteus spleen cell line, and the preservation number of the Pampus argenteus spleen cell line is CCTCC NO: C2025154.
[0006] The present application first provides a Pampus argenteus spleen cell line, which has the advantages of stable passage, high survival rate and strong sensitivity to Iridovirus.
[0007] Further, the second aspect of the present application provides a construction method of the Pampus argenteus spleen cell line in the first aspect, comprising the following steps:
[0008] S1: under sterile conditions, taking out the spleen tissue of Pampus argenteus, and rinsing the spleen tissue;
[0009] S2: disinfecting the spleen tissue rinsed in step S1;
[0010] S3: passing the spleen tissue after sterilization in step S2 through a cell screen to collect a cell suspension;
[0011] S4: centrifuging the cell suspension in step S3 to collect cells;
[0012] S5: culturing the cells collected in S4 using a first culture medium, removing the culture medium and non-adherent cells after the cells adhere to the wall, and continuing to culture the adherent cells using a second culture medium to obtain primary cells;
[0013] S6: subculturing the primary cells to obtain a spleen cell line of Pseudosciaena crocea.
[0014] Preferably, the reagent used for rinsing in step S1 is Dulbecco's phosphate buffered saline containing 100 IU / mL of penicillin, 100 µg / mL of streptomycin, 100 µg / mL of gentamicin and 3 µg / mL of soluble amphotericin B.
[0015] The present application uses Dulbecco's phosphate buffered saline containing antibiotics / antifungal agents as a rinsing solution, which can effectively eliminate common microorganisms (such as gram-positive bacteria and fungi) on the surface of the spleen tissue, prevent cross contamination in subsequent culture, and effectively ensure the initial cleanliness of the cells.
[0016] Preferably, the reagent used for sterilization in step S2 is DMEM / F-12 culture medium containing 100 IU / mL of penicillin, 100 µg / mL of streptomycin, 100 µg / mL of gentamicin and 3 µg / mL of soluble amphotericin B.
[0017] The present application uses DMEM / F-12 culture medium containing antibiotics / antifungal agents as a sterilization solution for sterilization, which further inactivates potential pathogens embedded in the tissue, and has sterilization ability far superior to PBS. At the same time, using DMEM / F-12 culture medium as a sterilization solution can effectively ensure cell viability and lay a foundation for the healthy growth of primary cells.
[0018] Preferably, the first culture medium is DMEM / F-12 culture medium containing 1% volume fetal bovine serum, 100 IU / mL of penicillin, 100 µg / mL of streptomycin and 100 µg / mL of gentamicin.
[0019] For Pseudosciaena crocea spleen cells, using a low serum culture medium for initial culture can avoid excessive growth and pollution spread caused by excessive nutrition. At the same time, culturing through a low serum culture medium can quickly screen cells with strong adhesion ability.
[0020] Preferably, the second culture medium is DMEM / F-12 culture medium containing 20% volume fetal bovine serum, 100 IU / mL of penicillin, 100 µg / mL of streptomycin and 100 µg / mL of gentamicin.
[0021] As preferred, the step of primary culture is: after the cells form a monolayer, the cells are digested by trypsin containing EDTA, then resuspended by a second culture medium and cultured in a flask, and the process is repeated for 5-10 times, and the primary culture is completed.
[0022] The primary culture of the application can quickly screen out cell lines with high activity, strong stability and good adhesion by repeating trypsin digestion-resuspension-culture.
[0023] As preferred, the subculture includes the following steps:
[0024] S61: after the primary cells form a monolayer, the cells are digested by trypsin containing EDTA, then the digested cells are subcultured by a third culture medium at a ratio of 1:2;
[0025] S62: after subculture for 25-35 generations, the cells are digested by trypsin containing EDTA, then the digested cells are subcultured by a fourth culture medium at a ratio of 1:2;
[0026] S63: after subculture for 95-105 generations, the cells are digested by trypsin containing EDTA, then the digested cells are subcultured by a fifth culture medium at a ratio of 1:2, and the silver pomfret spleen cell line is obtained.
[0027] As preferred, the third culture medium is DMEM / F-12 culture medium containing 15% volume fetal bovine serum, 50 IU / mL of blue and streptomycin;
[0028] The fourth culture medium is DMEM / F-12 culture medium containing 10% fetal bovine serum;
[0029] The fifth culture medium is DMEM / F-12 culture medium containing 5% fetal bovine serum.
[0030] In subculture, the application gradually reduces the serum content and antibiotic concentration in the culture medium, which improves the activity of the cells and reduces the detection background, facilitating subsequent virology and gene expression research.
[0031] Further, the third aspect of the application provides applications of the silver pomfret spleen cell line of the first aspect, and the applications include
[0032] Application I: the application of the silver pomfret spleen cell line in iridovirus culture;
[0033] and / or application II: the application of the silver pomfret spleen cell line in iridovirus immune detection;
[0034] and / or application III: the application of the silver pomfret spleen cell line in exogenous gene expression.
[0035] It should be noted that, for the purpose of non-disease diagnosis and treatment, the cell line provided by the present application can be used for research on fish iridovirus culture carrier, iridovirus pathogenesis and exogenous gene expression, and has wide application prospect.
[0036] Different from the prior art, the present application has the beneficial effects of:
[0037] 1. The silver pomfret spleen cell line provided by the present application can be cultured using 5% serum and antibiotic-free medium, which can effectively reduce the detection background in virology and cytology research.
[0038] 2. The silver pomfret spleen cell line provided by the present application is extremely sensitive to fish cell hypertrophic iridovirus, and obvious cytopathic effect can be observed after 2 days of inoculation, which solves the problem of virus diagnosis in aquaculture and provides a basis for the development of antiviral drugs. BRIEF DESCRIPTION OF DRAWINGS
[0039] Figure 1 It is a cell photo of primary culture in Example 1 of the present application;
[0040] Figure 2 It is a cell photo when subcultured for 30 generations in Example 1 of the present application;
[0041] Figure 3 It is a cell photo when subcultured for 100 generations in Example 1 of the present application;
[0042] Figure 4 It is a light microscope photo of the control group cells not infected with virus in Example 2 of the present application;
[0043] Figure 5 It is a light microscope photo of the cells infected with RSIV in Example 2 of the present application;
[0044] Figure 6 It is a light microscope photo of the cells infected with SDDV in Example 2 of the present application;
[0045] Figure 7 It is an electron microscope photo of the cells infected with RSIV in Example 2 of the present application;
[0046] Figure 8 It is an electron microscope photo of the cells infected with SDDV in Example 2 of the present application;
[0047] Figure 9 It is a light microscope photo of the large yellow croaker head kidney cell line not infected with virus in Comparative Example 1 of the present application;
[0048] Figure 10 It is a light microscope photo of the cytopathic effect of the large yellow croaker head kidney cell line infected with RSIV for 48 hours in Comparative Example 1 of the present application;
[0049] Figure 11 The light microscope photo of the cytopathic effect of the cell line of Pseudosciaena crocea head infected with SDDV for 48 hours in the present application comparative example 1.
[0050] BIOLOGICAL DEPOSIT
[0051] The present application relates to biological preservation, the name of the preserved culture: the spleen cell line YC-S01 of Pseudosciaena crocea, the preservation number: CCTCC NO: C2025154, the preservation time: May 15, 2025, the preservation organization: China Center for Type Culture Collection, the preservation address: Wuhan University, Wuhan, China. DETAILED DESCRIPTION
[0052] In order to make the above-mentioned objectives, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present application, and are not used to limit the parameter range described in the present application, and the reasonable changes derived therefrom are still within the protection scope of the claims of the present application.
[0053] It should be noted that the endpoints of the ranges and any values disclosed herein are not limited to the precise values recited. The ranges and values should be interpreted as being approximate, and the endpoints of the ranges of values should be considered to be open-ended. The endpoints of the ranges of values can be combined with one another to form one or more new ranges of values, which should be considered to be within the scope of the present application.
[0054] In order to promote the research of iridovirus, the specific embodiment of the present application provides a spleen cell line of Pseudosciaena crocea, and the preservation number of the spleen cell line of Pseudosciaena crocea is CCTCC NO: C2025154.
[0055] The construction method of the above-mentioned spleen cell line of Pseudosciaena crocea comprises the following steps:
[0056] S1: under sterile conditions, the spleen tissue of Pseudosciaena crocea is taken out, and the spleen tissue is rinsed;
[0057] S2: the rinsed spleen tissue in step S1 is disinfected;
[0058] S3: the disinfected spleen tissue in step S2 is passed through a cell screen, and a cell suspension is collected;
[0059] S4: the cell suspension in step S3 is centrifuged, and cells are collected;
[0060] S5: Incubate the cells collected in S4 at a constant temperature using a first culture medium, and after the cells adhere, remove the culture medium and non-adherent cells, and continue primary culture of the adherent cells using a second culture medium to obtain primary cells;
[0061] S6: Passage culture of the primary cells to obtain a silver pomfret spleen cell line.
[0062] More specifically, in step S1 of the above embodiment, the reagent used for rinsing is Dulbecco's phosphate buffered saline containing 100 IU / mL of penicillin, 100 µg / mL of streptomycin, 100 µg / mL of gentamicin and 3 µg / mL of soluble amphotericin B.
[0063] More specifically, in step S2 of the above embodiment, the reagent used for disinfection is DMEM / F-12 culture medium containing 100 IU / mL of penicillin, 100 µg / mL of streptomycin, 100 µg / mL of gentamicin and 3 µg / mL of soluble amphotericin B.
[0064] More specifically, the first culture medium is DMEM / F-12 culture medium containing 1% volume of fetal bovine serum, 100 IU / mL of penicillin, 100 µg / mL of streptomycin and 100 µg / mL of gentamicin.
[0065] More specifically, the second culture medium is DMEM / F-12 culture medium containing 20% volume of fetal bovine serum, 100 IU / mL of penicillin, 100 µg / mL of streptomycin and 100 µg / mL of gentamicin.
[0066] More specifically, in the above embodiment, the step of primary culture is as follows: after the cells form a monolayer, the cells are digested using trypsin containing EDTA, and then resuspended in the second culture medium and cultured in a flask, and the process is repeated 5-10 times to complete the primary culture.
[0067] More specifically, in the above embodiment, the passage culture includes the following steps:
[0068] S61: After the primary cells form a monolayer, the cells are digested using trypsin containing EDTA, and then 1:2 passage culture of the digested cells is performed using a third culture medium;
[0069] S62: After passage culture for 25-35 generations, the cells are digested using trypsin containing EDTA, and then 1:2 passage culture of the digested cells is performed using a fourth culture medium;
[0070] S63: After passage culture for 95-105 generations, the cells are digested using trypsin containing EDTA, and then 1:2 passage culture of the digested cells is performed using a fifth culture medium to obtain a silver pomfret spleen cell line.
[0071] More specifically, in the above-mentioned embodiments, the third culture medium is DMEM / F-12 medium containing 15% volume fetal bovine serum, 50 IU / mL of penicillin, and streptomycin;
[0072] The fourth culture medium is DMEM / F-12 medium containing 10% fetal bovine serum;
[0073] The fifth culture medium is DMEM / F-12 medium containing 5% fetal bovine serum.
[0074] The above-mentioned spleen cells of silver pomfret are sensitive to iridovirus cells and can be used for related research on iridovirus.
[0075] The technical solutions of the present application are further described below through specific examples. Unless otherwise defined, all terms, symbols and other scientific and technical terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. In some cases, terms having commonly understood meanings are defined herein for the sake of clarity or convenience. Such definitions should not be interpreted to mean that the commonly understood meanings are significantly different from the meanings defined herein. The technical methods described or referenced herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise specified, the use of commercially available kits and reagents, instruments is carried out according to the protocols and parameters provided by the manufacturers. Unless otherwise specified, the temperature of cell culture in the following examples is 26°C.
[0076] Example 1
[0077] YC-S01 cell construction
[0078] S1: Select five silver pomfrets with a body weight of about 60 g, anesthetize the silver pomfrets with 50 mg / L eugenol, and then place them on a clean bench. Open the abdominal cavity of the silver pomfret with scissors and forceps, carefully remove the spleen from the fish body, and then rinse the spleen with Dulbecco's phosphate buffered saline containing 100 IU / mL of penicillin, streptomycin, 100 µg / mL of gentamicin, and 3 µg / mL of soluble amphotericin B. The rinsing is performed for 3 times, each time for 5 min;
[0079] S2: Disinfect the spleen tissue rinsed in step S1 with DMEM / F-12 medium containing 100 IU / mL of penicillin, streptomycin, 100 µg / mL of gentamicin, and 3 µg / mL of soluble amphotericin B. The disinfection is performed for 30 min;
[0080] S3: Transfer the spleen tissue disinfected in step S2 into a new DMEM / F-12 medium containing 100 IU / mL of penicillin, streptomycin, 100 µg / mL of gentamicin, and 3 µg / mL of soluble amphotericin B. Cut the spleen and pass it through a 70 μm cell strainer to collect the cell suspension;
[0081] S4: centrifuge the cell suspension in step S3 at room temperature using a horizontal rotor centrifuge, collect the cells, the centrifugal speed is 1000-1200g, and the centrifugal time is 2-3min;
[0082] S5: use the first culture medium to culture the cells collected in S4 at 26℃ overnight, remove the culture medium and non-adherent cells, and continue to culture the adherent cells using the second culture medium to obtain primary cells, wherein the first culture medium is DMEM / F-12 culture medium containing 1% volume fetal bovine serum, 100IU / mL of penicillin, streptomycin, and 100µg / mL of gentamicin, the second culture medium contains 20% volume fetal bovine serum, 100IU / mL of penicillin, streptomycin, and 100µg / mL of gentamicin, and the primary culture step is: after the cells form a monolayer, use trypsin (0.25% trypsin, 0.05% EDTA) to digest the cells in the cell culture bottle, the digestion time is 20-30s, resuspend the digested cells with the second culture medium, and continue to culture in the original bottle, repeat 5-10 times, and the morphology of the primary cultured cells under an optical microscope is as shown in Figure 1 ;
[0083] S6: subculture the primary cells to obtain a silver pomfret spleen cell line, wherein the subculture includes the following steps:
[0084] S61: after the primary cells form a monolayer, use trypsin (0.25% trypsin, 0.05% EDTA) to digest the cells, and then use the third culture medium to subculture the digested cells at a ratio of 1:2, wherein the third culture medium is DMEM / F-12 culture medium containing 15% volume fetal bovine serum, 50IU / mL of penicillin, and streptomycin;
[0085] S62: after 30 generations of subculture, use trypsin (0.25% trypsin, 0.05% EDTA) to digest the cells, and then use the fourth culture medium to subculture the digested cells at a ratio of 1:2, wherein the fourth culture medium is DMEM / F-12 culture medium containing 10% fetal bovine serum, and the morphology of the cells at the 30th generation under an optical microscope is as shown in Figure 2 ;
[0086] S63: After 100 passages, the cells were digested with trypsin (0.25% trypsin, 0.05% EDTA), and then passaged 1:2 in medium V. Once medium V had no significant effect on cell proliferation rate and cell state, the silver pomfret spleen cell line YC-S01 was obtained. The medium V was DMEM / F-12 medium containing 5% fetal bovine serum. Cell morphology after 100 passages under a light microscope is shown below. Figure 3 As shown.
[0087] Depend on Figures 1 to 3 It can be seen that the cells provided by the present invention are in good condition in DMEM / F-12 medium containing 5% fetal bovine serum after 100 passages.
[0088] Example 2
[0089] Virus infection experiment
[0090] A1: Take one flask of YC-S01 cells in good growth condition, add trypsin (0.25% trypsin, 0.05% EDTA) to digest the cells, then add 5 mL of complete culture medium, and repeatedly pipette the bottom of the cell culture flask to disperse the cells and form a uniform cell suspension.
[0091] A2: Seed the cell suspension into 6-well plates, 2 mL per well, and incubate the culture plates in a 26°C cell culture incubator until the cells adhere to the wall at about 80%;
[0092] A3: Take out the red sea bream iridovirus (RSIV) and scale shedding virus (SDDV) viruses that were frozen at -80℃ and thaw them quickly in a 37℃ water bath.
[0093] A4: After centrifuging the thawed virus preservation solution at 4°C and 10000 g for 3-5 min, 100 µL of the supernatant was seeded into 6-well plates. The cytopathic effect was observed daily. Figure 4 These are uninfected control group cells. Figure 5 The cytopathic effect was observed 48 hours after YC-S01 infection with RSIV. Figure 6 The cytopathic effect was observed 48 h after YC-S01 infection with SDDV. Figures 2 to 4 It is evident that YC-S01 exhibits excellent sensitivity to both RSIV and SDDV, with cells showing cytopathic effects after inoculation, making it suitable for direct pathogen research.
[0094] A5: After culturing for 3 days, the cells were digested using trypsin (0.25% trypsin and 0.05% EDTA), centrifuged, the supernatant was removed, and the precipitate was sequentially washed with DMEM / F-12 without fetal bovine serum and Dulbecco's phosphate buffer, and then fixed in a PBS buffer containing 0.5% fetal bovine serum and 2.5% glutaraldehyde at 4°C overnight in the dark;
[0095] A6: After washing the fixed cells using a PB buffer, the cells were again fixed using osmic acid, and after 1.5 hours of fixation, the cells were again washed using a PB buffer;
[0096] A7: After washing, the cells were sequentially dehydrated using 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol, 100% ethanol and 100% acetone;
[0097] A8: The dehydrated cells were permeated using a first embedding liquid (embedding agent and acetone in a volume ratio of 1:2) for 1 hour, and then permeated using a second embedding liquid (embedding agent and acetone in a volume ratio of 2:1) for 1.5 hours, and then vacuum permeated with embedding agent overnight, and finally embedding agent was added to the sample and polymerized at 65°C for 48 hours;
[0098] A9: After polymerization, the sample was sectioned and the section was dyed using 2% uranyl acetate-ethanol solution, and the section was observed using a transmission electron microscope.
[0099] Figure 7 Fig. 2 shows the morphology and distribution of intracellular virus particles at different resolutions under an electron microscope after the YC-S01 was infected with RSIV, Figure 8 Fig. 3 shows the morphology and distribution of intracellular virus particles at different resolutions under an electron microscope after the YC-S01 was infected with SDDV. It can be seen that Figure 7 and Figure 8 It can be seen that the YC-S01 cell line provided by the application has been completely infected with two viruses, and can be used for subsequent pathogenic research.
[0100] Comparative Example 1
[0101] Cell line control test
[0102] The existing large yellow croaker head kidney cell line (LYC-hK) was used as a control cell line, and the test method was the same as that of Example 2. After 48 hours of culture, an optical microscope was used for observation, and the results are shown in Figures 9 to 11 Fig. 4, wherein Figure 9 Fig. 4a is a light microscope photograph of the large yellow croaker head kidney cell line without virus infection, Figure 10 Fig. 4b is the cytopathic effect of the large yellow croaker head kidney cell line after being infected with RSIV for 48 hours, Figure 11 Fig. 4c is the cytopathic effect of the large yellow croaker head kidney cell line after being infected with SDDV for 48 hours.
[0103] By Figures 9 to 11 It can be seen that the existing cell lines have very poor sensitivity to fish cell hypertrophic iridovirus, and no obvious cytopathic effect can be observed after 48 hours of infection, which also proves that the YC-S01 provided by the present application has very high sensitivity to fish cell hypertrophic iridovirus, and is an excellent carrier for fish iridovirus culture and research.
[0104] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited to this. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will fall within the protection scope of the present disclosure.
Claims
1. A silver pomfret spleen cell line, characterized in that, The preservation number of the silver pomfret spleen cell line is CCTCC NO: C2025154.
2. A method of constructing a silver pomfret spleen cell line as claimed in claim 1, wherein the method comprises the steps of: Comprising the following steps: S1: Under sterile conditions, the spleen tissue of the silver pomfret is taken out, and the spleen tissue is rinsed; S2: The spleen tissue rinsed in step S1 is disinfected; S3: The spleen tissue disinfected in step S2 is passed through a cell screen, and a cell suspension is collected; S4: The cell suspension in step S3 is centrifuged, and cells are collected; S5: The cells collected in S4 are incubated using a first culture medium, after the cells adhere, the culture medium and non-adherent cells are removed, and a second culture medium is used to continue primary culture of the adherent cells, and primary cells are obtained; S6: The primary cells are subcultured to obtain a silver pomfret spleen cell line.
3. The construction method of claim 2, wherein, In step S1, the rinsing reagent is D-PBS containing 100 IU / mL of penicillin, streptomycin, 100 µg / mL of gentamicin and 3 µg / mL of soluble amphotericin B.
4. The construction method of claim 2, wherein, In step S2, the disinfecting reagent is DMEM / F-12 medium containing 100 IU / mL of penicillin, streptomycin, 100 µg / mL of gentamicin and 3 µg / mL of soluble amphotericin B.
5. The construction method of claim 2, wherein, The first culture medium is DMEM / F-12 medium containing 1% volume fetal bovine serum, 100 IU / mL of penicillin, streptomycin, 100 µg / mL of gentamicin.
6. The construction method of claim 2, wherein, The second culture medium is DMEM / F-12 medium containing 20% volume fetal bovine serum, 100 IU / mL of penicillin, streptomycin, 100 µg / mL of gentamicin.
7. The construction method of claim 2, wherein, The step of primary culture is: after the cells form a monolayer, the cells are digested using trypsin containing EDTA, then resuspended with the second culture medium and cultured in a flask, and the primary culture is repeated 5-10 times.
8. The construction method of claim 2, wherein, The subculture includes the following steps: S61: After the primary cells form a monolayer, the cells are digested using trypsin containing EDTA, and the digested cells are subcultured at 1:2 using a third culture medium; S62: After 25-35 passages of subculture, the cells are digested using trypsin containing EDTA, and the digested cells are subcultured at 1:2 using a fourth culture medium; S63: After 95-105 passages of subculture, the cells are digested using trypsin containing EDTA, and the digested cells are subcultured at 1:2 using a fifth culture medium to obtain a silver pomfret spleen cell line.
9. The construction method of claim 8, characterized in that, The third culture medium is DMEM / F-12 medium containing 15% volume fetal bovine serum, 50 IU / mL of penicillin, streptomycin; The fourth culture medium is DMEM / F-12 medium containing 10% fetal bovine serum; The fifth culture medium is DMEM / F-12 medium containing 5% fetal bovine serum.
10. Use of the silver pomfret spleen cell line of claim 1, characterized in that, The applications include Application 1: The silver pomfret spleen cell line is used in iridovirus culture. And / or application two: the application of the silver pomfret spleen cell line in iridovirus immune detection; And / or application three: the application of the silver pomfret spleen cell line in exogenous gene expression.