Pseudosciaena crocea head kidney macrophage line as well as construction method and application thereof

Abstract

The invention provides a larimichthys crocea head kidney macrophage line as well as a construction method and application thereof, belongs to the technical field of cell engineering, and particularly provides the larimichthys crocea head kidney macrophage line which is named as LYC-hK and has the preservation number of CCTCC NO: C2025234. The larimichthys crocea head kidney macrophage line is constructed through the steps S1-S6, and the construction method of the larimichthys crocea head kidney macrophage line is applied to the field of cell engineering. The larimichthys crocea head kidney macrophage line provided by the invention can be used for research on pathogenic infection and pathogenic mechanism of larimichthys crocea, research on anti-infection immune response of larimichthys crocea and research on exogenous gene expression.

Description

Technical Field

[0001] This invention relates to the field of cell engineering technology, and more specifically, to a large yellow croaker head kidney macrophage cell line, its construction method, and its application. Background Technology

[0002] Large yellow croaker (Larimichthys crocea) is an important marine aquaculture fish in my country. In recent years, with the continuous expansion of aquaculture scale, various diseases have frequently occurred, especially "visceral white spot disease," which has become a major factor restricting the healthy development of large yellow croaker aquaculture. In the fish immune system, the head kidney is an important hematopoietic and immune organ, and macrophages, as important cells of innate immunity, play a crucial role in resisting the invasion of pathogenic microorganisms. Therefore, establishing a stable large yellow croaker head kidney macrophage cell line is of great significance for studying the pathogenesis and immune control of diseases in large yellow croaker.

[0003] Currently, there are several research reports on the isolation and culture of head kidney macrophages from large yellow croaker. Chinese invention patent application CN102533651B discloses a method for isolating and primary culturing head kidney macrophages from large yellow croaker. This method utilizes Percoll level centrifugation at a 31% / 45% gradient to isolate fish head kidney macrophages, which are then cultured at 22°C in L15 composite medium containing fetal bovine serum, penicillin, streptomycin, heparin, etc. The proportion of macrophages in the isolated cells is as high as 42%, and the purity of adherent large yellow croaker head kidney macrophages after overnight culture is over 90%. Chinese invention patent with publication number CN110295136A discloses a method for establishing a large yellow croaker head kidney macrophage cell line. The method selects high-purity primary head kidney macrophage cells from marine fish, large yellow croaker, and continuously culturees them at 28°C in DMEM / F12 containing 15% FBS. The medium is replaced by a method of replacing half of the medium, and the macrophages are passaged using PBS-EDTA-assisted trypsin digestion.

[0004] However, the existing technology still has the following problems: First, although there are primary culture methods for large yellow croaker head kidney macrophages, the cells cultured by these methods have limited survival time and are difficult to passage stably for a long time; second, the reported large yellow croaker head kidney cell lines are mainly fibroblasts, and there is a lack of macrophage cell lines specifically for studying diseases such as "visceral white spot disease" in large yellow croaker; third, the culture medium composition and culture conditions in the existing culture methods are not optimized enough, making it difficult to meet the requirements for long-term stable passage of large yellow croaker head kidney macrophages; fourth, there is a lack of standardized and preserved large yellow croaker head kidney macrophage cell lines, which limits the development of related research and the sharing of results. Therefore, it is urgent to establish a stable large yellow croaker head kidney macrophage cell line that can be passaged for a long time to provide an effective cell model for studying the pathogenic mechanism and immune control of diseases such as "visceral white spot disease" in large yellow croaker. Summary of the Invention

[0005] The purpose of this invention is to address the lack of cell lines in the existing technology specifically for studying the pathogenic factors and pathogenic mechanisms of "visceral white spot disease" in large yellow croaker, and to provide a large yellow croaker head kidney macrophage cell line for studying the pathogenic mechanisms and immune control of fish pathogenic bacteria.

[0006] To solve the above-mentioned technical problems, the first aspect of the present invention provides a large yellow croaker head kidney macrophage cell line, which is named LYC-hK and has the accession number CCTCC NO: C2025234.

[0007] A second aspect of this invention provides a method for constructing the large yellow croaker head kidney macrophage cell line described in the first aspect, comprising the following steps: S1: Under aseptic conditions, the head kidney tissue of the large yellow croaker was removed and rinsed with rinsing solution; S2: After rinsing, disinfect the head and kidney tissue with the first disinfectant; S3: After disinfection, remove the connective tissue from the head kidney tissue and pass the head kidney tissue through a sieve to obtain a head kidney cell suspension; S4: Centrifuge the kidney cell suspension and collect the cell pellet; S5: Culture the cell pellet in the first culture medium. After the cells adhere, remove the culture medium and non-adherent cells, and then use the second culture medium to culture the adherent cells in primary culture to obtain primary cells. S6: Primary cells were passaged to obtain the head kidney macrophage cell line from large yellow croaker.

[0008] Preferably, the rinsing solution is Duchenne phosphate buffer containing 100 IU / mL penicillin-streptomycin, 100 µg / mL gentamicin, and 3 µg / mL soluble amphotericin B.

[0009] Preferably, the first culture medium is DMEM / F-12 medium containing 0.5% fetal bovine serum, 100 IU / mL penicillin and streptomycin, and 100 µg / mL gentamicin.

[0010] Preferably, the second culture medium is DMEM / F-12 medium containing 20% ​​fetal bovine serum, 100 IU / mL penicillin, streptomycin, and 100 µg / mL gentamicin.

[0011] Preferably, step S6 includes the following steps: S61: Primary cells are cultured using a second culture medium. After the primary cells form a monolayer, they are digested with trypsin containing EDTA. The digested cells are then passaged 1:2 using a third culture medium. S62: After passage to the 25th-35th generation, the cells were digested with trypsin containing EDTA, and the digested cells were passaged 1:2 using the fourth medium. S63: After passage to the 95th-105th generation, the cells were digested with trypsin containing EDTA, and the digested cells were passaged 1:2 in the fifth medium to obtain the head kidney macrophage cell line of large yellow croaker.

[0012] Preferably, the third culture medium is DMEM / F-12 medium containing 13%~15% fetal bovine serum and 50 IU / mL penicillin and streptomycin.

[0013] Preferably, the fourth culture medium is DMEM / F-12 medium containing 8% to 10% by volume fetal bovine serum. Preferably, the fifth culture medium is DMEM / F-12 medium containing 5% fetal bovine serum.

[0014] A third aspect of the present invention provides an application of the large yellow croaker head kidney macrophage cell line described in the first aspect, the application comprising: Application 1: The application of the large yellow croaker head kidney macrophage cell line in the pathogen infection and pathogenesis mechanism of large yellow croaker; And / or Application 2: Application of the large yellow croaker head kidney macrophage cell line in the anti-infection immune response of large yellow croaker; And / or Application 3: Application of the large yellow croaker head kidney macrophage cell line in exogenous gene expression.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The large yellow croaker head kidney macrophage cell line provided by this invention can be cultured in a medium containing 5% serum and free of antibiotics, which can effectively reduce the detection background in cell biology research; 2. The large yellow croaker head kidney macrophage cell line provided by this invention has the characteristics of stable passage and high survival rate, strong respiratory burst activity and phagocytic activity against pathogenic bacteria, and can be used as a cell model for studying the virulence factors and pathogenic mechanisms of pathogenic bacteria, providing a basis for the development of antibacterial drugs and immunomodulators for large yellow croaker. 3. The large yellow croaker head kidney macrophage cell line provided by this invention is specifically designed to address the research needs of "visceral white spot disease" in large yellow croaker, filling the gap in cell models in this field and providing an important tool for studying the pathogenic mechanism and immune control of pathogenic bacteria in large yellow croaker. Attached Figure Description

[0016] Figure 1 This is a photograph of the morphology of primary cultured cells under an optical microscope in Example 1 of the present invention; Figure 2These are cell morphology photographs taken under an optical microscope when the cells have been passaged to the 30th generation in Example 1 of this invention. Figure 3 These are cell morphology photographs taken under an optical microscope when the cells have been passaged to the 100th generation in Example 1 of this invention. Figure 4 These are cell morphology photographs taken under an optical microscope when the cells have been passaged to the 100th generation in Example 1 of this invention. Figure 5 This is a confocal microscope image of the cellular respiratory burst of LYC-hK cells induced by LPS and PMA in Example 2 of the present invention; Figure 6 This is an ultrastructural photograph of normal LYC-hK cells in Example 3 of the present invention; Figure 7 This is an ultrastructural image of LYC-hK cells infected with Nocardia amberjack in Example 3 of the present invention, showing that the LYC-hK cells contain multiple Nocardia amberjack cells; Figure 8 This is an ultrastructural image of LYC-hK cells infected with Pseudomonas amberjack in Example 3 of the present invention, showing that LYC-hK cells contain multiple Nocardia amberjack cells; Figure 9 This is a statistical chart showing the survival of Nocardia amberjack and Pseudomonas spp. in Example 4 of the present invention after invading LYC-hK cells for 6 h, 12 h and 24 h. Figure 10 Electrophoresis diagram showing the expression of β-actin, CD68, CD163, CD209, CD302, mpeg1, CD86, CD11b, and CSF1Ra in LYC-hK cells in Example 5 of this invention.

[0017] Biological Preservation Statement: This application relates to biological preservation. The culture name is: Large Yellow Croaker Head Kidney Macrophage Cell Line LYC-hK, preservation number: CCTCC NO: C2025234, preservation date: August 6, 2025, preservation institution: China Center for Type Culture Collection, preservation address: Wuhan University, Wuhan, China. Detailed Implementation

[0018] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the present invention.

[0019] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] To promote research on the pathogenesis, vaccine products, and immune detection of white spot disease in fish viscera, this invention provides a large yellow croaker head kidney macrophage cell line, named LYC-hK, with accession number CCTCC NO: C2025234.

[0021] The method for constructing the large yellow croaker head kidney macrophage cell line in the above embodiments includes the following steps: S1: Under aseptic conditions, the head kidney tissue of the large yellow croaker was removed and rinsed with rinsing solution; S2: After rinsing, disinfect the head and kidney tissue with the first disinfectant; S3: After disinfection, remove the connective tissue from the head kidney tissue and pass the head kidney tissue through a sieve to obtain a head kidney cell suspension; S4: Centrifuge the kidney cell suspension and collect the cell pellet; S5: Culture the cell pellet in the first culture medium. After the cells adhere, remove the culture medium and non-adherent cells, and then use the second culture medium to culture the adherent cells in primary culture to obtain primary cells. S61: Primary cells are cultured using a second culture medium. After the primary cells form a monolayer, they are digested with trypsin containing EDTA. The digested cells are then passaged 1:2 using a third culture medium. S62: After passage to the 25th-35th generation, the cells were digested with trypsin containing EDTA, and the digested cells were passaged 1:2 using the fourth medium. S63: After passage to the 95th-105th generation, the cells were digested with trypsin containing EDTA, and the digested cells were passaged 1:2 in the fifth medium to obtain the head kidney macrophage cell line of large yellow croaker.

[0022] In the above embodiments, the rinsing solution is Duchenne phosphate buffer containing 100 IU / mL penicillin and streptomycin, 100 µg / mL gentamicin, and 3 µg / mL soluble amphotericin B.

[0023] In the above embodiments, the first culture medium is DMEM / F-12 medium containing 0.5% fetal bovine serum, 100 IU / mL penicillin and streptomycin, and 100 µg / mL gentamicin.

[0024] In the above embodiments, the second culture medium is DMEM / F-12 medium containing 20% ​​fetal bovine serum, 100 IU / mL penicillin, streptomycin, and 100 µg / mL gentamicin.

[0025] In the above embodiments, the third culture medium is DMEM / F-12 medium containing 13%~15% volume fetal bovine serum and 50 IU / mL penicillin and streptomycin.

[0026] In the above embodiments, the fourth culture medium is DMEM / F-12 medium containing 8% to 10% volume fetal bovine serum.

[0027] In the above embodiments, the fifth culture medium is DMEM / F-12 medium containing 5% fetal bovine serum.

[0028] The specific embodiments of the present invention also include an application of the aforementioned large yellow croaker head kidney macrophage cell line, the application comprising: Application 1: The application of the large yellow croaker head kidney macrophage cell line in the pathogen infection and pathogenesis mechanism of large yellow croaker; And / or Application 2: Application of the large yellow croaker head kidney macrophage cell line in the anti-infection immune response of large yellow croaker; And / or Application 3: Application of the large yellow croaker head kidney macrophage cell line in exogenous gene expression.

[0029] The technical solutions of the present invention are further described below through specific embodiments. Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. In some cases, terms with a conventional understanding are limited herein for clarification or ease of reference, and such limitations should not be construed as indicating a significant difference from the conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and have been adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters. Unless otherwise stated, the cell culture temperature in the following examples is 26°C.

[0030] Example 1 Construction of LYC-hK cells S1: Six healthy large yellow croakers weighing approximately 30g were selected. After anesthetizing with 50mg / L eugenol, the head and kidney tissues of the large yellow croakers were removed under sterile conditions. The head and kidney tissues were rinsed with Duchenne phosphate buffer containing 100IU / mL penicillin-streptomycin, 100µg / mL gentamicin, and 3µg / mL soluble amphotericin B. The rinsing was repeated three times, for 5 minutes each time. S2: After rinsing, the head kidney tissue was disinfected with DMEM / F-12 medium containing 100 IU / mL penicillin and streptomycin, 100 µg / mL gentamicin and 3 µg / mL soluble amphotericin B for 30 min. S3: After disinfection, remove the connective tissue from the head kidney tissue and pass the head kidney tissue through a 70μm sieve to obtain a head kidney cell suspension; S4: Centrifuge the kidney cell suspension using a centrifuge head at a speed of 1000-1200g for 2-3 minutes, and collect the cell pellet. S5: At 26℃, the cell pellet was cultured in DMEM / F-12 medium containing 0.5% fetal bovine serum, 100 IU / mL penicillin, streptomycin, and 100 µg / mL gentamicin. After cell attachment, the medium and non-attached cells were removed, and the adherent cells were cultured in DMEM / F-12 medium containing 20% ​​fetal bovine serum, 100 IU / mL penicillin, streptomycin, and 100 µg / mL gentamicin to obtain primary cells. The cell morphology in the primary culture was as follows. Figure 1 As shown; S61: Primary cells were cultured in DMEM / F-12 medium containing 20% ​​fetal bovine serum, 100 IU / mL penicillin and streptomycin, and 100 µg / mL gentamicin. After the primary cells formed a monolayer, they were digested with trypsin containing EDTA and passaged 1:2 in DMEM / F-12 medium containing 115% fetal bovine serum and 50 IU / mL penicillin and streptomycin. S62: After passage 30, cells were digested with trypsin containing EDTA and then passaged 1:2 in DMEM / F-12 medium containing 8%–10% fetal bovine serum. Cell morphology at passage 30 under an optical microscope is as follows. Figure 2 As shown; S63: After passage 100, cells were digested with trypsin containing EDTA and then passaged 1:2 in DMEM / F-12 medium containing 5% fetal bovine serum to obtain the large yellow croaker head kidney macrophage cell line, named LYC-hK. Cell morphology after passage 100 under an optical microscope is shown below. Figure 3 , Figure 4 As shown.

[0031] Depend on Figures 1-4 It can be seen that the large yellow croaker head kidney macrophage cell line provided by the present invention can still grow well in DMEM / F-12 medium with 5% fetal bovine serum after 100 passages.

[0032] Example 2 Respiratory burst experiment Follow these steps to conduct the experiment: A1: Take one flask of LYC-hK 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. A2: Seed the cell suspension into 6-well plates containing cell spreaders, 2 mL per well, and incubate the culture plate in a 26°C cell culture incubator until the cells adhere to the plate at about 80% of the plate. A3: The cells in A2 were co-incubated with lipopolysaccharide (LPS) at a final concentration of 2 μg / mL or phorbol 12-myristate 13-acetate (PMA) at a final concentration of 1 μg / mL for 6 h. A4: Add DCFH-DA (2,7-dichlorofuorescindiacetate) to A3 cells at a final concentration of 10µM and incubate the cells for 20 min; A5: Aspirate the culture medium and gently wash the cells with serum-free culture medium. Repeat this process 3 times. A6: The cell slides were removed and observed under a laser confocal microscope with an excitation wavelength of 488nm.

[0033] Experimental results are as follows Figure 5 As shown, by Figure 5 It is evident that LYC-hK cells, after induction with LPS or PMA, exhibit a stronger respiratory burst capacity compared to the control group. This can be used for research on related pathogen infections and pathogenic mechanisms.

[0034] Example 3 LYC-hK phagocytosis experiment Follow these steps to conduct the experiment: B1: Take one flask of LYC-hK 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. B2: 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 are about 80% attached. B3: The bacterial concentrations of Nocardia amberjack and Pseudomonas stolonifera in the late logarithmic growth phase were adjusted to approximately 1×10⁻⁶ using DF-12 medium. 8 CFU / mL, take 1 mL of bacterial solution and add it to B2 cells to make its infection coefficient approximately 50:1; B4: After infection in a 26℃ incubator for 6 hours, the culture medium was aspirated, and the cells were gently washed with serum-free culture medium. This process was repeated 3 times. B5: After digesting B4 cells with trypsin (trypsin 0.25%, EDTA 0.05%), centrifuge, remove supernatant, wash the precipitate with DMEM / F-12 without fetal bovine serum and DuPont phosphate buffer, and fix overnight at 4°C in the dark with PBS buffer containing 0.5% fetal bovine serum and 2.5% glutaraldehyde. B6: After washing the fixed cells with PB buffer, add osmium tetroxide to refix the cells. After fixing for 1.5 hours, wash the cells again with PB buffer. B7: After washing, the cells were dehydrated in sequence using 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol, 100% ethanol and 100% acetone. B8: The process involves permeating dehydrated cells with the first embedding solution (embedding agent to acetone volume ratio 1:2) for 1 hour, then permeating the sample with the second embedding solution (embedding agent to acetone volume ratio 2:1) for 1.5 hours, then permeating the sample under vacuum overnight with the embedding agent, and finally adding the embedding agent to the sample and polymerizing at 65°C for 48 hours. B9: After polymerization, the sample sections were stained with 2% uranium acetate-ethanol solution and observed using transmission electron microscopy.

[0035] The ultrastructure of normal LYC-hK cells, such as Figure 6 As shown, the ultrastructure of LYC-hK cells infected with Nocardia amberjack is as follows: Figure 7 As shown, the ultrastructure of LYC-hK cells infected with *Pseudomonas aeruginosa* is as follows: Figure 8 As shown, comparison Figures 6-8 It is evident that the LYC-hK cells provided by this invention can phagocytose Nocardia amberjack and Pseudomonas amberjackus, and can be used for subsequent research on the pathogenic factors and pathogenic mechanisms of these two bacterial pathogens.

[0036] Example 4 Study on the survival of pathogenic bacteria in LYC-hK cells Follow these steps to conduct the experiment: C1: Take one flask of LYC-hK 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. C2: Seed the cell suspension into 12-well plates, 1 mL per well, and incubate the culture plates in a 26°C cell culture incubator until the cells are about 80% attached. C3: The bacterial concentrations of Nocardia amberjack and Pseudomonas stolonifera in the late logarithmic growth phase were adjusted to approximately 1×10⁻⁶ using DF-12 medium. 8 CFU / mL, take 0.5 mL of bacterial culture and add it to C2 cells to make its infection coefficient approximately 50:1; C4: After incubating the cells in a 26℃ incubator for 2 hours, aspirate the culture medium and gently wash the cells with serum-free culture medium. Repeat this process 3 times. C5: Add 1 mL of DMEM / F-12 medium containing 150 µg / mL kanamycin sulfate and treat for 1.5 h; C6: After sterilization, remove the culture medium, wash twice with sterile PBS, and add fresh culture medium containing 6.25 μg / mL. C7: C6 cells were placed in a 26°C incubator and cultured for 6 h, 12 h and 24 h. C8: Discard the C7 cell culture supernatant and wash twice with sterile PBS; C9: Add 1 mL of 50 mM Tris-HCl containing 0.5% Triton X-100 to each well to lyse cells for 10 min; C10: After repeated pipetting, C9 cells were transferred into 1.5 mL centrifuge tubes and serially diluted 10 times with sterile PBS. Samples of different dilutions were spread on TSA plates and incubated at 28°C for 2-7 days. Bacterial colonies in the plates were counted, and the intracellular survival rates of Nocardia amberjack and Pseudomonas stolonifera in LYC-hK cells were calculated.

[0037] Figure 9 The survival rates of Nocardia amberjack and Pseudomonas spp. invading LYC-hK cells at 6 h, 12 h, and 24 h were determined by [data missing]. Figure 9 It is evident that the number of bacteria gradually decreases over time after invading LYC-hK cells, indicating that LYC-hK cells have good bactericidal ability.

[0038] Example 5 Molecular marker expression experiment in LYC-hK cells Conduct the experiment according to the following steps: D1: Take one bottle of LYC-hK cells in good growth condition and extract total RNA from the cells using an RNA extraction kit; D2: The RNA obtained in D1 was reverse transcribed using a reverse transcription kit to obtain cDNA; D3: Using the cDNA obtained in D2 as a template, β-actin, CD68, CD163, CD209, CD302, mpeg1, CD86, CD11b, and CSF1Ra were amplified by PCR. The amplification products were then detected by gel electrophoresis.

[0039] Gel electrophoresis results as follows Figure 10 As shown, by Figure 10 It can be seen that genes such as β-actin, CD68, CD163, CD209, CD302, mpeg1, CD86, CD11b, and CSF1Ra can be detected in LYC-hK cells, indicating that this cell line has macrophage characteristics.

[0040] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A large yellow croaker head kidney macrophage cell line, characterized in that, The large yellow croaker head kidney macrophage cell line was named LYC-hK, with accession number CCTCC NO: C2025234.

2. A method for constructing the large yellow croaker head kidney macrophage cell line according to claim 1, characterized in that, Includes the following steps: S1: Under aseptic conditions, the head kidney tissue of the large yellow croaker was removed and rinsed with rinsing solution; S2: After rinsing, disinfect the head and kidney tissue with the first disinfectant; S3: After disinfection, remove the connective tissue from the head kidney tissue and pass the head kidney tissue through a sieve to obtain a head kidney cell suspension; S4: Centrifuge the kidney cell suspension and collect the cell pellet; S5: Culture the cell pellet in the first culture medium. After the cells adhere, remove the culture medium and non-adherent cells, and then use the second culture medium to culture the adherent cells in primary culture to obtain primary cells. S6: Primary cells were passaged to obtain the head kidney macrophage cell line from large yellow croaker.

3. The construction method as described in claim 2, characterized in that, The rinsing solution is Duchenne phosphate buffer containing 100 IU / mL penicillin-streptomycin, 100 µg / mL gentamicin, and 3 µg / mL soluble amphotericin B.

4. The construction method as described in claim 2, characterized in that, The first culture medium was DMEM / F-12 medium containing 0.5% fetal bovine serum, 100 IU / mL penicillin and streptomycin, and 100 µg / mL gentamicin.

5. The construction method as described in claim 2, characterized in that, The second culture medium was DMEM / F-12 medium containing 20% ​​fetal bovine serum, 100 IU / mL penicillin, streptomycin, and 100 µg / mL gentamicin.

6. The construction method as described in claim 2, characterized in that, Step S6 includes the following steps: S61: Primary cells are cultured using a second culture medium. After the primary cells form a monolayer, they are digested with trypsin containing EDTA. The digested cells are then passaged 1:2 using a third culture medium. S62: After passage to the 25th-35th generation, the cells were digested with trypsin containing EDTA, and the digested cells were passaged 1:2 using the fourth medium. S63: After passage to the 95th-105th generation, the cells were digested with trypsin containing EDTA, and the digested cells were passaged 1:2 in the fifth medium to obtain the head kidney macrophage cell line of large yellow croaker.

7. The construction method as described in claim 6, characterized in that, The third culture medium is DMEM / F-12 medium containing 13%~15% fetal bovine serum and 50 IU / mL penicillin and streptomycin.

8. The construction method as described in claim 6, characterized in that, The fourth culture medium is DMEM / F-12 medium containing 8%~10% fetal bovine serum.

9. The construction method as described in claim 6, characterized in that, The fifth culture medium is DMEM / F-12 medium containing 5% fetal bovine serum.

10. An application of the large yellow croaker head kidney macrophage cell line according to claim 1, characterized in that, The applications include Application 1: The application of the large yellow croaker head kidney macrophage cell line in the pathogen infection and pathogenesis mechanism of large yellow croaker; And / or Application 2: Application of the large yellow croaker head kidney macrophage cell line in the anti-infection immune response of large yellow croaker; And / or Application 3: Application of the large yellow croaker head kidney macrophage cell line in exogenous gene expression.

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