Stable passage cell line derived from sebastes schlegeli fin ray tissue

Abstract

The invention provides a stable passage cell line SSFin cell derived from fin ray tissue of sebastes schlegeli, and the stable passage cell line SSFin cell can be used for related research on disease-resistant immunity of sebastes schlegeli. The preservation number of the sebastes schlegeli fin ray tissue cell line SSFin provided by the invention is CCTCC (China Center For Type Culture Collection) NO: C202371. The stable passage cell line derived from fin ray tissue of sebastes schlegeli provided by the invention has sensitivity to pathogenic bacteria infection, and can be used as a sensitive cell for aquatic etiology and immunology research.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to a stable cell line derived from the fin ray tissue of Scorpionfish xu Sebastes schlegelii The invention discloses a SSFin cell line, or SSFin cells for short, and its establishment method and application. The cell line can be applied to aquatic pathogenicity and immunology research. Background Art

[0002] my country is a major aquaculture country. In recent years, in response to the demand for aquatic products brought about by population growth and economic progress, the scale of aquaculture in my country has grown rapidly. With the increasing degree of aquaculture intensification, diseases frequently occur, which restricts the green and healthy development of the aquaculture industry. Therefore, clarifying the anti-disease immune mechanism of aquatic animals and the infection and pathogenic mechanism of aquatic pathogens can provide basic data for the prevention and control of aquatic diseases and provide ideas for the design of anti-disease treatment strategies, which is of great significance to the green and healthy development of the aquaculture industry.

[0003] Xu's flathead scorpionfish Sebastes schlegelii ), belongs to the genus Sebastidae of the order Perciformes ( Sebastes ), with its tender meat, delicious taste, strong reproductive capacity, and natural overwintering, it is an important species for cage farming and resource enhancement and release in northern my country. However, the aquaculture of Xu's flatfish is endangered by bacterial, viral, and parasitic diseases, such as Vibrio harveyi ( Vibrio harveyi )、Vibrio rotifer( Vibrio rotiferianus ) can cause skin ulcers and bleeding on the surface and internal organs of fish. Lymphocystis disease virus (LCDV) can cause a large number of irregular nodules on the surface of fish. Philometridae can parasitize on the gill covers, pectoral fins, oral walls and other parts of the fish, causing skin damage and then bacterial infection in the damaged parts. Studies have shown that the direct exposure of the fish surface to the water environment is one of the ways for various pathogens to invade. However, the cell lines of S. schrenkiana that have been established for etiology and immunology research only include myoblast cell lines derived from muscle tissue and cell lines (SSI) derived from intestinal tissue. There is a lack of cell lines derived from surface tissues, which restricts the study of the pathogenic mechanism of pathogens infecting S. schrenkiana through the surface of the body. Summary of the invention

[0004] The purpose of the present invention is to provide a stable cell line SSFin cells (SSFin cells) derived from the fin ray tissue of S. schrenkiana, which can be used for the disease resistance and immunity related research of S. schrenkiana.

[0005] The Sebastes schlegelii fin tissue cell line SSFin ( Sebastes schlegelii L. fincell SSFin) provided by the present invention was deposited on April 25, 2023 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C202371.

[0006] The present invention also provides a use of the cell line SSFin, which is an application in etiology and immunology; More specifically, the use is an application as a bacterial infection model; The bacterium is a pathogen capable of infecting Sebastes schlegelii; As a specific record of an embodiment, the pathogen is Edwardsiella piscicida.

[0007] The stable passaging cell line derived from the Sebastes schlegelii fin tissue provided by the present invention is sensitive to pathogen infection and can be used as a sensitive cell for aquaculture etiology and immunology research. Description of the Drawings

[0008] Figure 1 : Schematic diagram of the establishment process of the SSFin cell line; wherein, A: Juvenile Sebastes schlegelii used for cell line establishment; B: Primary cells migrated from fin tissue and the morphological characteristics of passage cells from the 2nd to the 80th generation; C and D: Karyotype analysis of SSFin cells; Figure 2 : Growth curve diagram of SSFin cells under different temperatures and different serum concentrations; wherein, A: Growth curves of SSFin cells under the culture conditions of 20°C, 24°C and 28°C; B: Growth curves of SSFin cells under the serum concentrations of 5%, 10%, 15% and 20%; Figure 3 : Diagram of the reproduction of Edwardsiella piscicida in SSFin cells; wherein, A: After infecting SSFin cells with Edwardsiella piscicida containing GFP plasmid, the proliferation of bacteria in the cells was observed by laser confocal microscopy at 0 h, 2 h, 6 h and 10 h; B: After infecting SSFin cells with Edwardsiella piscicida containing GFP plasmid for 10 h, the proliferation of bacteria in the cells was observed by laser confocal microscopy (oil immersion lens); C: At different time points after infecting SSFin cells with Edwardsiella piscicida, the reproduction of bacteria in the cells was detected by plating; Figure 4 : Statistical analysis diagram of differential genes after infecting SSFin cells with Edwardsiella piscicida; Figure 5 : GO functional enrichment analysis diagram of differential genes after infecting SSFin cells with Edwardsiella piscicida; Figure 6 : KEGG signaling pathway enrichment analysis of differentially expressed genes after Edwardsiella piscicida infection of SSFin cells; Figure 7 : Production of intracellular reactive oxygen species after Edwardsiella piscicida infection of SSFin cells; Specific implementation mode

[0009] The present invention provides a cell line (SSFin cells) derived from fin ray tissue, which is highly sensitive to infection by Edwardsiella piscicida ( Edwardsiella piscicida ) and can mount an immune response to the infection. The present invention is of great significance for promoting the research on the disease resistance immunity and pathogenic mechanism of Sebastes schlegelii, and has good application prospects.

[0010] SSFin cells were infected with Edwardsiella piscicida, and the proliferation of the pathogen in the cells was analyzed by laser confocal microscopy observation and plate coating detection methods. The changes in gene expression and the production of reactive oxygen species after the cells responded to pathogen infection were analyzed by transcriptome sequencing and intracellular reactive oxygen species detection to determine the sensitivity of SSFin to Edwardsiella piscicida infection.

[0011] The present invention also provides the application of a cell line (SSFin) derived from the fin ray tissue of Sebastes schlegelii in the fields of etiology and immunology, etc.

[0012] The present invention will be further described below with reference to the accompanying drawings and examples, but it does not limit the scope of the rights of the present invention. The technical solutions described in the present invention are all conventional solutions in the art unless otherwise specified; the reagents or materials are all from commercial channels unless otherwise specified.

[0013] Example 1: Establishment and detection of SSFin cell line To construct a fin ray cell line of Sebastes schlegelii, the surface of juvenile Sebastes schlegelii was disinfected with 70% alcohol, and the fin rays were cut off. After soaking in 70% alcohol for 1 - 2 seconds, they were quickly transferred to a sterile PBS solution, washed twice, and then transferred to DMEM (Dulbecco's Modified Eagle, Gibco, USA) medium. The fin rays were cut into pieces of 1 mm 3The fin ray pieces were transferred to a 12-well cell culture plate and adhered to the wall for 2 hours. Then, DMEM medium (with penicillin, streptomycin and 20% fetal bovine serum) was added and cultured in a 24°C incubator. After the tissue pieces migrated out of the wells and the cells filled the wells, trypsin was added to collect the digested cells and transferred to a new cell culture plate. After the cells filled the wells, they were subcultured at a ratio of 1:2. After the cell line was stably subcultured for 50 generations, subsequent experiments were carried out. In addition, the stably subcultured fin ray cells were sent to the Wuhan University Collection Center for preservation. Since the surface of the fish body is one of the invasion pathways for various pathogens, the currently established Xu's flat scorpionfish cell lines only include myoblast cell lines (myoblast cell lines) derived from muscle tissue and cell lines (SSI) derived from intestinal tissue, and lack cell lines derived from surface tissues. Therefore, the Xu's flat scorpionfish fin ray cell line constructed in this application has tissue source specificity and novelty and has preservation value.

[0014] Karyotype analysis of SSFin cells showed that the number of chromosomes was 48, which was consistent with the number of chromosomes of the flatfish. Growth curve analysis of SSFin cells at different temperatures and serum concentrations revealed that the optimal temperature was 24°C and the optimal serum concentration was 20%, and subsequent experiments also used these culture conditions.

[0015] Example 2: Sensitivity test of SSFin cells to Edwardsiella piscicida In order to study the sensitivity of SSFin cells to Edwardsiella piscicida, Edwardsiella piscicida transformed with pFPV25.1-EGFP plasmid (the bacteria appear green under a fluorescence microscope) was used to infect SSFin cells. The reproduction of bacteria in cells at different infection time points was observed by laser confocal microscopy. It was found that bacteria reproduced in SSFin cells 2 h after infection, and the bacterial reproduction increased with the increase of infection time.

[0016] At 10 hours of infection, it can be clearly seen under the oil microscope that a large number of bacterial cells are aggregated in a single SSFin cell. After the cells were lysed at different infection time points and plated for colony counting, it was found that the number of bacteria continued to increase with the increase of infection time.

[0017] These results collectively indicate that SSFin cells are sensitive to Edwardsiella piscicola, that the bacteria can successfully infect cells and proliferate intracellularly, and that the number of bacterial proliferation increases with infection time.

[0018] Example 3: Response of SSFin cells to Edwardsiella piscicida infection Cells with an MOI of 20 were used to infect SSFin, and the cells were collected after 10 h for transcriptome sequencing. Compared with the uninfected cells, the gene expression levels in the bacteria-infected cells changed significantly. Among them, 369 genes were upregulated and 441 genes were downregulated. GO (Gene Ontology) functional enrichment analysis found that the genes with significant expression changes were involved in a variety of biological processes, including: signaling receptor binding, receptor ligand activity, transporter activity, etc.; KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling pathway enrichment analysis found that the genes with expression changes were involved in multiple signaling pathways, including: cytokine-cytokine receptor interaction, cell adhesion molecules, melanogenesis, etc. In addition, the intracellular reactive oxygen species content in SSFin cells after infection with Edwardsiella piscicida was measured, and it was found that a large amount of reactive oxygen species was produced. Since reactive oxygen species are lethal to bacteria, the production of reactive oxygen species by SSFin may be its resistance mechanism to bacterial infection. The above results indicate that SSFin cells can respond to the infection of Edwardsiella piscicida and can be applied to the research of etiology and immunology.

Claims

1. A fin ray tissue cell line of Scorpionfish xu, characterized in that: The deposit number of the cell line is CCTCC NO: C202371.

2. Use of the cell line according to claim 1 in pathogenesis and immunology.

3. The use according to claim 2, characterized in that The application described is that of a bacterial infection model.

4. The use according to claim 3, characterized in that The bacteria are pathogens that can infect the flathead mullet.

5. The use according to claim 4, characterized in that The pathogen is Edwardsiella piscicidal.

6. Use of the cell line according to claim 1 in preparing products for studying bacterial infection of Scorpionfish.

7. A product for studying bacterial infection of Scorpionfish, characterized in that: The preparation uses the cell line described in claim 1 as an infection model.

8. A method for studying bacterial infection of Scorpionfish for non-disease diagnosis and treatment purposes, characterized in that: The method uses the cell line of claim 1 as an infection model.

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