Spleen cell line of ptereleotris punctatus and application thereof

Abstract

The application discloses a Plectropomus leopardus spleen cell line and application thereof. Plectropomus leopardus The spleen cell line PLS has a preservation number of GDMCC No: 68131. The PLS is obtained through primary culture and multiple subculture, has a fast cell growth speed, has been stably subcultured for more than 60 generations, has good activity after cryopreservation, and lays a foundation for Plectropomus leopardus gene germplasm resource preservation. Meanwhile, the PLS is sensitive to fish nervous necrosis virus and iridovirus, can be applied to exogenous gene function research, fish virus isolation, virus infection pathogenesis, virus host interaction and virus in-vitro infection model, and provides a high-efficiency amplification system for Plectropomus leopardus virus vaccine preparation.

Description

Technical Field

[0001] This invention belongs to the field of marine fish cell culture biotechnology, and more specifically, relates to a spleen cell line of the leopard gill sea bass and its application. Background Technology

[0002] Leopard-gill spiny perch ( Plectropomus leopardus The Leopard Sea Bass (also known as the Eastern Star Grouper) is a species of fish belonging to the genus *Leopard* in the family Scutiidae, order Perciformes. It is one of the important marine aquaculture species in southern my country. Due to its vibrant coloration, tender and delicious flesh, rich nutrition, and high economic value, the Leopard Sea Bass is highly sought after by consumers and enjoys a broad market demand. Currently, artificial breeding of the Leopard Sea Bass is mainly carried out in indoor recirculating aquaculture systems and marine cages in Hainan. In recent years, the rapid development of the Leopard Sea Bass aquaculture industry and the stress on the breeding environment have led to frequent outbreaks of various diseases during the breeding process, resulting in significant economic losses and posing a major challenge to the healthy and sustainable development of the Leopard Sea Bass aquaculture industry. Among these, viral diseases are a significant infectious disease affecting Leopard Sea Bass aquaculture. Red-spotted grouper nervous necrosis virus (RGNNV) and leopard coral grouper iridovirus (LCGIV) are important pathogens during the juvenile stage of Leopard Sea Bass, with extremely high mortality rates after infection. However, effective treatments and prevention measures are currently lacking.

[0003] Fish cells, as an in vitro culture system, are widely used for the isolation and identification of fish viruses, the functional analysis of host genes, the study of molecular mechanisms of viral infection and replication, and the preparation of viral vaccines due to their good stability and controllable conditions. To date, few cell lines have been established specifically for the leopard-gill sea bass, including only brain, head kidney, snout, intestinal, and gill cell lines. A spleen cell line for the leopard-gill sea bass has not yet been reported. Therefore, to better study the pathogenic mechanism of leopard-gill sea bass viruses, screen effective antiviral drugs, or establish new prevention and control measures, there is an urgent need for cell lines sensitive to leopard-gill sea bass virus pathogens such as neuronecrosis virus and iridovirus as experimental support materials. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a spleen cell line of the leopard-gill sea bass ( Plectropomus leopardus Spleen cell line (PLS).

[0005] A second objective of this invention is to provide the application of the leopard gill spiny perch spleen cell line PLS.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: A spleen cell line of leopard gill spiny perch ( Plectropomus leopardus The Spleen cell line (PLS) was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 22, 2026, with accession number GDMCC No: 68131.

[0007] This invention obtained the PLS cell line from the spleen tissue of *Scourgeaurus leopardii* through primary culture and multiple passage selection. This cell line exhibits rapid growth, has been stably passaged for over 60 generations, and maintains good viability after cryopreservation, laying the foundation for the preservation of *Scourgeaurus leopardii* germplasm resources. It was deposited on April 22, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC No. 68131.

[0008] Further transfection experiments were conducted on the PLS (leopard gill spiny perch) spleen cell line to transfect exogenous genes. The results showed that the exogenous genes were efficiently expressed in PLS cells, suggesting that the PLS cell line can be used for in vitro studies of viral or host gene function. Virus sensitivity testing of PLS ​​cells showed that they were sensitive to at least two viruses: Nervous Necrosis Virus (NNV) and / or Iridovirus (IV). The viruses could infect and replicate well in PLS cells and induce host cells to initiate an immune response to inhibit viral replication after viral infection. This indicates that PLS can be used to study the immune defense response of host cells during viral infection and for the development of viral vaccines. Simultaneously, the virus could successfully proliferate in PLS cells, indicating that PLS cells can serve as an in vitro viral infection model for studying virus isolation in fish, viral pathogenic mechanisms, and virus-host interactions.

[0009] Therefore, the present invention also provides the application of the leopard-gill spiny perch spleen cell line PLS in the following aspects: Application of the leopard-gill spiny perch spleen cell line PLS in expressing exogenous genes.

[0010] Application of the spleen cell line PLS of the leopard gill sea bass as a host cell for studying marine fish viruses, namely neuronecrosis virus and / or iridovirus.

[0011] Application of the leopard-gill spiny perch spleen cell line PLS in the culture and / or detection of marine fish viruses for non-disease diagnostic purposes, wherein the marine fish viruses are neuronecrosis virus and / or iridovirus.

[0012] Application of the spleen cell line PLS of the leopard gill sea bass in the isolation of marine fish viruses, wherein the marine fish viruses are neuronecrosis virus and / or iridovirus.

[0013] Application of the spleen cell line PLS of the leopard gill sea bass in the development of inactivated vaccines for marine fish viruses, wherein the marine fish viruses are neuronecrosis virus and / or iridovirus.

[0014] Furthermore, the inactivated marine fish virus vaccine uses virus fluid that has been passaged up to 5 times as the virus seed.

[0015] Application of the leopard gill spiny perch spleen cell line PLS as an in vitro infection model for fish viruses, wherein the marine fish viruses are neuronecrosis virus and / or iridovirus.

[0016] Furthermore, the neuronecrosis virus is the red-spotted grouper neuronecrosis virus (RGNNV).

[0017] Furthermore, the iridovirus is leopard gill iridovirus LCGIV.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a spleen cell line of the leopard-gill spiny perch ( Plectropomus leopardus The leopard-gill spiny perch (PLS) spleen cell line was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on April 22, 2026, with accession number GDMCC No: 68131. The morphology of this PLS cell line is primarily fibroblast-like, and it has been stably passaged for over 60 generations. After thawing at -80℃, it exhibits good condition, laying the foundation for the preservation of leopard-gill spiny perch genetic resources. Furthermore, this leopard-gill spiny perch spleen cell line is sensitive to fish neuronecrosis virus and iridovirus, and can be used for research on the molecular mechanisms of fish viral pathogenesis, virus-host cell interactions, in vitro viral infection models, exogenous gene expression and function, and the preparation of iridovirus LCGIV inactivated vaccines. Attached Figure Description

[0019] Figure 1 This image shows the morphological characteristics of PLS ​​cells from the spleen of the leopard-gill spiny perch in Example 1 of this invention. Figure 1 In the image, A represents the cell morphology of PLS ​​cells in the 5th generation; B represents the cell morphology of PLS ​​cells in the 55th generation.

[0020] Figure 2 The results of PCR amplification using leopard-gill spiny perch specific primers were used to identify the species origin of PLS ​​cells in Example 2 of this invention.

[0021] Figure 3 This describes the detection of exogenous gene GFP expression in PLS cells in Example 3 of the present invention. Specifically, Figure 3 In the image, A shows the cell morphology of PLS ​​cells under a phase-contrast microscope; B shows the green fluorescence expressed by PLS cells transfected with pEGFP-C1.

[0022] Figure 4 In Example 4 of this invention, the sensitivity of PLS ​​cells to RGNNV is measured. The black arrows represent vacuoles caused by viral infection, and the red arrows represent intercellular spaces caused by viral infection.

[0023] Figure 5 This is a sensitivity test of PLS ​​cells to LCGIV in Example 4 of the present invention. The black arrows in the diagram represent cell swelling and rounding caused by LCGIV infection; the red arrows represent plaques produced by LCGIV infection.

[0024] Figure 6 This refers to the transcriptional changes of viral genes (CP and RdRp) after RGNNV infection with PLS in Example 4 of the present invention.

[0025] Figure 7 This refers to the transcriptional changes of viral genes (MCP and MMP) after LCGIV infection with PLS in Example 4 of this invention.

[0026] Figure 8 This refers to the changes in the expression of host genes, including the pro-inflammatory factor TNFα and the interferon-related gene ISG15, after RGNNV infection with PLS in Example 4 of the present invention.

[0027] Figure 9 This illustrates the proliferation of RGNNV in PLS cells in Example 4 of this invention. The red arrows represent RGNNV virus particles. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] Example 1: Establishment of the Leopard Gill Spinach Spleen Cell Line PLS (1) In the following examples, the fish selected were cultured leopard gill spiny perch with a body length of 6 cm. Plectropomus leopardus ).

[0031] (2) The Leibovitz's-15 (L-15) basal medium, HEPES, dual antibiotics (penicillin-streptomycin), 0.25% trypsin-EDTA digestion solution, and fetal bovine serum used in the following examples were products of GIBCO. All cell culture media used in this example were supplemented with NaCl. The specific formulation was L-15 basal medium with 0.266% NaCl and 5 mM HEPES by mass, adjusted to pH 7.4, filtered through a 0.22 μm filter, dispensed, and stored at 4°C for later use. Different concentrations of fetal bovine serum or dual antibiotics (penicillin-streptomycin) were added as needed when using passaged cells.

[0032] Tissue washing culture medium: L-15 cell culture medium with added penicillin (400 IU / mL penicillin and 400 μg / mL streptomycin).

[0033] Primary cell culture medium: Fetal bovine serum was added to the rinsing medium at a volume ratio of 20%. The culture medium used for the first 10 passages of cells was primary cell culture medium.

[0034] Subculture medium: After the cells have reached passage 10, the culture medium used for subculturing the cells is L-15 cell culture medium with 10% serum and penicillin-streptomycin (concentrations of 100 IU / mL penicillin and 100 μg / mL streptomycin, respectively).

[0035] (3) Primary culture Under aseptic conditions, the anesthetized fish fry were rinsed with 75% alcohol, and their spleen tissue was dissected and collected. The tissue was then rinsed three times with rinsing culture medium, 5 minutes each time. Next, the tissue pieces were thoroughly chopped with a sterile scalpel, and 0.5 mL of 0.25% trypsin-EDTA digestion solution was added for digestion at room temperature for 10 minutes. The digestion reaction was terminated by adding 4 mL of primary cell culture medium, and the tissue was transferred to a 25 cm² incubator. 2 The cells were cultured in a 28°C incubator, with half of the culture medium replaced every 4 days. Cell growth was observed and recorded daily. After approximately 12 days, the primary cells had covered about 80% of the bottom of the flask, at which point subculturing began.

[0036] (4) Subculture At the initial passage, the culture medium from the primary cell culture flask was collected and transferred to a sterile culture flask. The monolayer of cells was digested at room temperature with 1 mL of 0.25% trypsin-EDTA digestion solution. Then, 5 mL of fresh passaged cell culture medium and 5 mL of the collected primary cell culture medium were mixed, and the cells were resuspended in the mixture. The mixture was then aliquoted into two flasks at a 1:1 volume ratio and cultured at 28°C. Subcultures were performed every 4 days in the initial stage. At the 10th passage, the cell culture medium was changed to L15 medium containing 10% fetal bovine serum and containing double antibiotics (100 IU / mL penicillin and 100 μg / mL streptomycin). Subcultures were performed every 4 days. At this stage, the spleen cells of the leopard-gill sea bass exhibited mainly fibroblast-like and epithelial-like morphology. When subculturing from the 11th passage, once the cells adhered for 10 minutes, the culture medium was immediately replaced with fresh medium, leaving only the rapidly adhering cells. After 5 passages using the same method, the cell morphology became relatively uniform. Figure 1 Currently, this cell line has been stably passaged for over 60 generations and has been named the spleen cell line of the leopard-gill spiny perch. Plectropomus leopardus Spleen cell line (PLS).

[0037] (5) Cryopreservation and resuscitation capabilities of PLS ​​cells Take the cell suspension from the 50th generation in step (4), centrifuge at 1000 rpm to collect the cells, and resuspend them in 1 mL of cryopreservation solution pre-cooled at 4℃ (L-15 culture medium containing 20% ​​fetal bovine serum and 10% dimethyl sulfoxide by volume). Then place the cryopreservation tubes in a cryopreservation box and place them in an ultra-low temperature freezer at -80℃ overnight. The next day, transfer them to liquid nitrogen for long-term storage.

[0038] Cells were thawed after one month of cryopreservation. Cryopreservation tubes were quickly removed from liquid nitrogen and placed in a 37°C water bath for thawing. The cell pellet was collected by centrifugation, resuspended in 5 mL of passaged cell culture medium, and transferred to a culture flask for incubation at 28°C. Cell adhesion and growth were observed. No significant differences were observed in the morphology and proliferation capacity of the thawed cells compared to before cryopreservation.

[0039] (6) Identification of species origin of PLS ​​cells Cell pellets from passage 10 were collected by centrifugation after digestion. RNA was extracted using the Ce1l Total RNA Isolation Kit (FOREGENE, product number RE-03113), and reverse transcribed using the ReverTraAce® qPCR RT Kit (TOYOBO, product number FSQ-101B). Specific PCR amplification of the elongation factor 1alpha (EF1α) gene was then performed using the primer sequences EF1α-F:TGGTCGTCACCTTTGCTCC (SEQ ID No. 1) and EF1α-R:CGGCCACTGTCTGCCTCAT (SEQ ID No. 2). PCR amplification conditions were: 95 °C, 3 min; 94 °C, 25 s, 60 °C, 25 s, 72 °C, 15 s, 35 cycles; 72 °C, 5 min; 16 °C, 30 min. The obtained PCR products were subjected to electrophoresis and DNA sequencing.

[0040] The results showed that a specific band of the EF1α gene of the leopard-gill spiny perch could be amplified, and the sequencing results analysis showed that the obtained sequence (SEQ ID No. 3: TTGGTCGTCACCTTTGCTCCCCCTGCCCTGACCACTGAGGTGAAGTCTGTGGAGATGCACCACGAATCTCTGCCAGAGGCTGTCCCTGGTGACAATGTCGGTTTCAACATCAAGAACGTGTCAGTCAAGGAAATCCGTCGTGGATACGTGGCTGGCGACAGCAAGAACGACCCACCCAAGGGAGCTGACAACTTCAATGCTCAGGTCATCATCCTGAACCACCCTGGCCAGATTAACGCTGGTTAC) The sequence GCCCCTGTGCTGGATTGCCACACCGCTCACATTGCCTGCAAGTTCACCGAACTGATCGAGAAAATTGACCGTCGTTCTGGCAAGAAGCTTGAGGACGCTCCCAAGTTTGTCAAGTCTGGAGACGCCGCCATTGTCAAACTGGTCCCACAGAAGCCCATGGTTGTGGAGCCCTTCTCCAACTACCCTCCCCTCGGTCGTTTTGCTGTGCGTGACATGAGG) is identical to the EF1α sequence of the leopard-gill spiny perch, suggesting that the PLS cells originate from the leopard-gill spiny perch. Figure 2 ).

[0041] The spleen cell line of the leopard gill spiny perch ( Plectropomus leopardus Spleen cell line (PLS) was deposited on April 22, 2026 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC No: 68131.

[0042] Example 2: Successful expression of exogenous genes in PLS cells The PLS cells from passage 50 in Example 1 were passaged and seeded in 24-well cell culture plates and cultured for 18 hours before transfection experiments. The transfection reagent was Lipofectamine 2000 (Invitrogen), and the pEGFP-C1 plasmid was transfected according to the reagent instructions. Forty-eight hours after transfection, the distribution of green fluorescence within the cells was observed under a fluorescence microscope.

[0043] The results are as follows Figure 3 As shown, a strong green fluorescent signal was observed in PLS cells, indicating that the pEGFP-C1 plasmid was successfully transfected into PLS cells, demonstrating that the foreign gene can be efficiently expressed in PLS cells, suggesting that the PLS cell line can be used to study the function of viral or host genes in vitro.

[0044] Example 3 Virus susceptibility testing of PLS ​​cell lines 1. Cytopathic effect (CPE) caused by viral infection PLS cells from passage 50 of Example 1 were seeded into 24-well culture plates and cultured overnight before being inoculated with viruses, including Red Spotted Grouper Neuronecrosis Virus (RGNNV) and Leopard Gill Iridoid Virus (LCGIV), both with a Multiplicity of Infection (MOI) of 2. The CPE process induced by the virus infection was observed under a microscope at different time points and photographed for recording.

[0045] The results are as follows Figure 4 As shown, after RGNNV infection of PLS ​​cells, vacuolation began to appear in the cells at 24 hours. With prolonged infection time, vacuolation became increasingly severe at 48 hours, and gaps appeared due to cell rounding. In contrast, after LCGIV infection of PLS ​​cells (… Figure 5 Cells began to round out after 24 hours, and the number of rounded cells increased significantly after 48 hours, with obvious intercellular spaces (CPE) appearing. The control cells (Mock group) showed no significant change in cell morphology. This indicates that PLS is sensitive to at least two viruses, RGNNV and LCGIV.

[0046] 2. Detection of viral gene transcription after viral infection of cells Following the method described in step 1 of Example 3, RGNNV and LCGIV were infected respectively. Cells were collected at different time points after viral infection (LCGIV: 24 and 48 h; RGNNV: 24 and 48 h), and total RNA was extracted from the cells using the Ce1l Total RNA Isolation Kit (FOREGENE, product number RE-03113). cDNA synthesis was performed on the obtained total RNA using the ReverTraAce® qPCR RT Kit (TOYOBO, product number FSQ-101B). Furthermore, the transcriptional levels of viral genes, including CP, RdRp, MCP, and MMP, were detected using a 2×polarsignal® SYBR Green qPCR Mix Kit (MIKX, product number MKG802-10) on an AppliedBiosystems QuantStudio 5 real-time PCR instrument. The conditions for quantitative real-time PCR were as follows: 95℃ for 1 min; (95℃ for 5 s; 60℃ for 10 s; 72℃ for 15 s) × 40 (40 cycles). β-actin was used as an internal reference gene, and the relative expression level of the target gene was calculated using the 2-ΔΔCt method. The primer sequences used are shown in Table 1.

[0047] Table 1 Primer sequences used for quantitative virus detection

[0048] The results are as follows Figure 6 As shown, transcription of CP and RdRp genes could be detected at both 24h and 48h after RGNNV infection of PLS, and the transcription levels of both genes were significantly higher at 48h than at 24h; similarly, after LCGIV infection of PLS ​​cells ( Figure 7 MCP and MMP gene transcription could be detected at both 24h and 48h, and the transcription levels of both genes were significantly higher at 48h than at 24h, indicating that viral gene transcription of RGNNV and LCGIV increased with the extension of infection time after PLS infection, suggesting that the virus can infect and replicate well in PLS cells.

[0049] 3. Transcription of host immune genes after RGNNV infection of cells Following the method described in step 2 of Example 3, RNA was extracted from RGNNV-infected PLS cells, reverse transcribed, and full-length cDNA was obtained. A quantitative real-time PCR reaction system was then prepared using a 2×polarsignal® SYBR Green qPCR Mix kit (MIKX, catalog number MKG802-10) to measure the transcriptional levels of host immune genes, including TNFα and ISG15, on an Applied Biosystems QuantStudio 5 quantitative PCR instrument. The quantitative PCR reaction conditions were as follows: 95℃ for 1 min; (95℃ for 5 s; 60℃ for 10 s; 72℃ for 15 s) × 40 (40 cycles). β-actin was used as an internal reference gene, and the relative expression level of the target gene was calculated using the 2-ΔΔCt method. The primer sequences used are shown in Table 2.

[0050] Table 2 Primer sequences used for quantitative detection of host immune genes

[0051] The results are as follows Figure 8 As shown, compared with the control group (Mock), the transcriptional levels of TNFα and ISG15 in PLS cells were significantly increased at both 24 h and 24 h after RGNNV infection, suggesting that host cells initiate an immune response to suppress viral replication after viral infection. These results indicate that PLS can be used to study the immune defense response of host cells during viral infection.

[0052] Example 4: Proliferation of RGNNV in PLS cells PLS cells from passage 50 of Example 1 were infected with RGNNV following step 1 of Example 3. Cells were collected 48 h after viral infection, centrifuged, and the pellet was fixed with 2.5% glutaraldehyde at 4°C for 1 h. After discarding the fixative, the cells were washed three times with PBS for 5 min each time; then fixed with 1% (v / v) osmium tetroxide at 4°C for 1 h; followed by stepwise dehydration with ethanol gradients (50%, 70%, 80%, 90%, 100%, 100% v / v) for 10 min each time. The cells were then embedded in Epon 812 epoxy resin. Finally, after preparing ultrathin sections, the samples were stained with 2% uranium acetate and lead citrate for 1 h each, and the cell and virus structures were observed and photographed under a transmission electron microscope (Talos L120C, Thermo Fisher Scientific).

[0053] The results are as follows Figure 9As shown, vacuolar structures formed by a single membrane were observed in RGNNV-infected cells. These vacuolar structures contained a large number of uniformly distributed viral particles. Furthermore, scattered viral particles were also observed in the cytoplasm, indicating that RGNNV virus can successfully proliferate in PLS cells. In conclusion, PLS cells can serve as an in vitro viral infection model for studying virus isolation in fish, viral pathogenic mechanisms, and virus-host interactions.

[0054] 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 within the protection scope of the present invention.

Claims

1. A spleen cell line of a leopard-gill spiny perch ( Plectropomus leopardus Spleen cell line (PLS), characterized by, The cell line was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 22, 2026, with accession number GDMCC No: 68131.

2. The application of the spleen cell line of the leopard gill sea bass according to claim 1 in expressing exogenous genes.

3. The application of the spleen cell line of the leopard-gill sea bass according to claim 1 as a host cell for studying marine fish viruses, characterized in that, The marine fish virus is a neuronecrosis virus and / or an iridovirus.

4. The application of the spleen cell line of the leopard-gill sea bass according to claim 1 in the culture and / or detection of viruses in marine fish for non-disease diagnostic purposes, characterized in that... The marine fish virus is a neuronecrosis virus and / or an iridovirus.

5. The application of the spleen cell line of the leopard-gill sea bass according to claim 1 in the isolation of viruses from marine fish, characterized in that, The marine fish virus is a neuronecrosis virus and / or an iridovirus.

6. The application of the spleen cell line of the leopard-gill sea bass according to claim 1 in the development of inactivated virus vaccines for marine fish, characterized in that, The marine fish virus is a neuronecrosis virus and / or an iridovirus.

7. The application according to claim 6, characterized in that, The inactivated vaccine for marine fish viruses uses viral fluid that has been passaged up to 5 times as the virus seed.

8. The application of the spleen cell line of the leopard-gill sea bass according to claim 1 as an in vitro virus infection model for marine fish, characterized in that, The marine fish virus is a neuronecrosis virus and / or an iridovirus.

9. The application according to any one of claims 3 to 8, characterized in that, The nerve necrosis virus mentioned is the red-spotted grouper nerve necrosis virus (RGNNV).

10. The application according to any one of claims 3 to 8, characterized in that, The iris virus in question is leopard gill iris virus LCGIV.

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