Allogynogenetic silver crucian carp swim bladder cell line and application thereof

Abstract

The invention relates to a carassius auratus gibelio swim bladder cell line which is preserved in the China Center for Type Culture Collection on December 10, 2024, and the preservation number is CCTCC NO: C202476. The immortal cell line GiSB is constructed from the allogynogenetic prussian carp swim bladder tissue, and the cell line is mainly composed of fibroblast-like cells, is uniform in cell morphology and good in growth state, is stable in passage at present, can be stored in a cryopreservation manner, and can be used for corresponding scientific research and application. The allogynogenetic prussian carp swim bladder cell line provided by the invention can efficiently express exogenous genes, and provides an ideal research tool for gene function analysis, exogenous protein expression, vaccine preparation and the like. The carassius auratus gibelio swim bladder cell line provided by the invention is sensitive to various aquatic viruses, especially sensitive to important viral pathogen carassius auratus gibelio herpes viruses of carassius auratus gibelio, provides a good experimental material for separating and identifying carassius auratus gibelio viruses, and can also be applied to research on virus-host cell interaction.

Description

Technical Field

[0001] The present invention relates to the field of aquatic animal cells, and more particularly, to a fibroblast-like swim bladder cell line of Carassius auratus gibelio var. and its application. Background Art

[0002] Carassius auratus gibelio var. is one of the important freshwater aquaculture varieties in China, and can reproduce offspring through gynogenesis, sexual reproduction and hybrid reproduction. However, aquatic diseases have always been one of the main challenges faced by freshwater aquaculture. Carassius auratus herpesvirus (CaHV) belongs to the genus Cyprinivirus of the family Alloherpesviridae and is the key pathogen known to cause hematopoietic necrosis in crucian carp and its variant Carassius auratus gibelio var.

[0003] Cell lines are valuable experimental materials in life science research because they have good homogeneity, which provides a basis for the consistency and reproducibility of experimental results. Cell lines are widely used in various fields, including virology, environmental toxicology, cell biology, gene expression research, genetics and genomics, and are also considered the gold standard for virus isolation and identification. Currently established Carassius auratus gibelio var. cell lines include the caudal fin cell line, brain cell line and skin cell line, which to a certain extent limit the research on Carassius auratus gibelio var. virus diseases.

[0004] So far, at least more than 800 freshwater and marine fish cell lines have been successfully established and widely used in many research fields such as fish immunology, ecological toxicology and aquatic virology. Currently, there is no report on fibroblast-like swim bladder cell lines derived from Carassius auratus gibelio var. at home and abroad. Summary of the Invention

[0005] The inventor created a Carassius auratus gibelio swim bladder cell line GiSB during work. It was deposited at the China Center for Type Culture Collection (CCTCC) on December 10, 2024, with the deposit number: CCTCC NO: C202476. GiSB is composed of cells with a fibroblast-like morphology, and its karyotype is 3n = 156, accounting for 46% of the number of chromosomes in the observed mitotic phases, which is consistent with the number of chromosomes in Carassius auratus gibelio. The doubling time of this cell line for subculture is 3 - 5 days. This cell line is susceptible to various aquatic animal viruses such as Carassius auratus herpesvirus (CaHV), Andrias davidianus ranavirus (ADRV), Rana gryliovirus (RGV), Grass carp reovirus (GCRV), Paralichthys olivaceus rhabdovirus (PORV), and Scophthalmus maximus rhabdovirus (SMRV), and can be genetically manipulated, providing new experimental materials for subsequent research on fish virology and immunology.

[0006] The present invention also provides the application of the above-mentioned Carassius auratus gibelio swim bladder cell line in protein expression. Since GiSB can be genetically manipulated and foreign genes can be effectively expressed in the cells, GiSB can be used for protein expression, production of protein products, preparation of vaccines, etc.

[0007] Since GiSB is susceptible to a variety of aquatic animal viruses, the present invention also provides the application of the above-mentioned Carassius auratus gibelio swim bladder cell line in the isolation or culture of aquatic animal viruses. These susceptible viruses can be isolated or cultured from water bodies or virus-carrying animals to provide corresponding biological products to meet the needs of production or research. GiSB is particularly sensitive to the important viral pathogen Carassius auratus herpesvirus of Carassius auratus gibelio, providing good experimental materials for the isolation and identification of Carassius auratus gibelio viruses, and can also be applied to the research on virus-host cell interactions.

[0008] In addition, the present invention also provides the application of the above-mentioned Carassius auratus gibelio swim bladder cell line in detecting aquatic animal viruses in water bodies.

[0009] The present invention also provides a method for detecting aquatic animal viruses, including the following steps:

[0010] S1: Filter and sterilize the water sample to be tested or homogenize the animal tissue, and then add it to the culture environment of the above-mentioned Carassius auratus gibelio swim bladder cell line;

[0011] S2: Observe the characteristic changes of the Carassius auratus gibelio swim bladder cell line cultured in S1.

[0012] In a specific embodiment, the characteristic change includes a change in cell morphology. After inoculation with the virus for 1 - 12 h, obvious pathological changes can occur in the cell morphology of GiSB, including cell rounding, shrinkage, and detachment, etc. Therefore, the change in cell morphology can be used as one of the evaluation indicators for the presence of aquatic animal viruses in the test sample.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1) The present invention constructs an immortalized cell line GiSB from the swim bladder tissue of Carassius gibelio var. CASIII. This cell line is mainly composed of fibroblast - like cells, with uniform cell morphology, good growth state, stable passage at present, can be cryopreserved, and can be used for corresponding scientific research and applications.

[0015] 2) The swim bladder cell line of Carassius gibelio var. CASIII provided by the present invention can efficiently express foreign genes, providing an ideal research tool for gene function analysis, foreign protein expression, and vaccine preparation, etc.

[0016] 3) The swim bladder cell line of Carassius gibelio var. CASIII provided by the present invention is sensitive to a variety of aquatic viruses, especially sensitive to the important viral pathogen Cyprinid herpesvirus 2 of Carassius gibelio var. CASIII, providing good experimental materials for the isolation and identification of Carassius gibelio var. CASIII viruses, and can also be applied to the research on virus - host cell interaction.

[0017] Preservation of the cell line

[0018] The swim bladder cell line of Carassius gibelio var. CASIII mentioned in the present invention was deposited on December 10, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is: CCTCC NO: C202476, and the taxonomic name is the swim bladder cell line GiSB of Carassius gibelio var. CASIII, and the Latin scientific name is Carassius gibelio swimbladder cell GiSB. Description of the drawings

[0019] Figure 1 It is a micrograph of the swim bladder cell line GiSB of Carassius gibelio var. CASIII, where A: The first - generation swim bladder cells of Carassius gibelio var. CASIII; B: The tenth - generation swim bladder cells of Carassius gibelio var. CASIII; C: The thirtieth - generation swim bladder cells of Carassius gibelio var. CASIII; C: The sixtieth - generation swim bladder cells of Carassius gibelio var. CASIII, bar = 100 μm.

[0020] Figure 2 It shows the effects of different temperature conditions on the growth of swim bladder cells of Carassius gibelio var. CASIII (16 °C, 25 °C, and 30 °C).

[0021] Figure 3Karyotype analysis of the swim bladder cell line GiSB of allogynogenetic crucian carp. Among them, A: Karyotype analysis diagram of GiSB cells, bar = 200μm; B: Chromosome number distribution diagram of 100 GiSB cells.

[0022] Figure 4 Detection of the susceptibility of the swim bladder cell line GiSB of allogynogenetic crucian carp to CaHV. Among them, A is the micrograph of the swim bladder cell line GiSB of allogynogenetic crucian carp at different days after inoculation with CaHV, bar = 100μm; B is the PCR result diagram for detecting the infection of GiSB by CaHV at different days by amplifying the CaHV MCP gene; C is the GiSB cells infected with CaHV observed by electron microscopy, bar = 2μm and bar = 500nm.

[0023] Figure 5 Detection of the susceptibility of the swim bladder cell line GiSB of allogynogenetic crucian carp to other aquatic viruses. Among them, A is the micrograph after inoculation with different aquatic animal viruses, bar = 100μm; B is the detection of the infection situation by PCR amplification of the corresponding virus gene.

[0024] Figure 6 Fluorescence micrographs of the swim bladder cell line GiSB of allogynogenetic crucian carp at 24h and 48h after transfection with the EGFP gene. EGFP is green fluorescent protein, bar = 200μm. Detailed implementation mode

[0025] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0026] 1. Construction of the swim bladder cell line GiSB of allogynogenetic crucian carp

[0027] (1) Select a healthy A+ strain allogynogenetic crucian carp about 10 cm in length with excellent growth status. After cutting the gill arch to bleed, wipe the body surface stains with an alcohol cotton ball, soak it in medical alcohol for 15 - 30 s, and dissect and remove the entire swim bladder part along the lateral line under sterile conditions, and transfer it to a PBS solution containing penicillin-streptomycin double antibody (1000 U / ml) with a final concentration of 10%, soak it 3 times, 30 min each time, and shake it upside down during the period to remove surface bacteria and blood cells.

[0028] (2) Cut the processed swim bladder tissue into tissue blocks of 1 mm 3 in size, and transfer it to 25 cm 2In a cell culture flask. After culturing in an inverted position at 25 °C for 4 h, add it to L-15 medium containing 20% FBS and 2% penicillin-streptomycin (the final concentration is 100 μg / ml streptomycin and 100 U / ml penicillin), and continue to culture statically at 25 °C. Observe the cell migration situation every day, and replace 50% of the complete medium every 3 - 5 days until the migrated cells cover the entire bottom of the flask.

[0029] (3) When the cells migrate out of the tissue and form a monolayer of cells covering the bottom of the flask, aspirate the old medium, digest with 1.5 ml of 0.25% trypsin for 2 min. After the cells become round and form single cells and detach from the cell culture flask, add the complete medium described in (2) to stop digestion and pipette to disperse the cell clumps, and transfer them to a new cell culture flask at a ratio of 1:2. After passage to the 20th generation, reduce the FBS content in the complete medium to 10 - 15%, and perform subculture in the same way.

[0030] After passage to the 60th generation, the GiSB cell line of Carassius auratus gibelio swim bladder is obtained. The cell morphology is as Figure 1 shown, mainly in the form of fibroblast-like cells. After repeated passage and screening, a stably passaged GiSB cell line of Carassius auratus gibelio swim bladder is finally obtained.

[0031] 2. Cryopreservation and resuscitation of GiSB

[0032] (1) Cryopreservation of Carassius auratus gibelio swim bladder cells: Prepare a cryopreservation solution of L-15:FBS:DMSO = 7:2:1, collect cells in good growth state and resuspend them in the cryopreservation solution, transfer to a 1.8 ml cryopreservation tube, make marks and place it in a programmable cooling box at -80 °C overnight, then transfer to liquid nitrogen for storage;

[0033] (2) Resuscitation of Carassius auratus gibelio swim bladder cells: Take out the Carassius auratus gibelio swim bladder cells from liquid nitrogen, quickly place them in a 37 °C water bath, gently shake until the ice completely melts, transfer the cell suspension to a 15 ml centrifuge tube, add 5 ml of complete medium, let it stand for 2 min, then centrifuge at 800 rpm for 10 min. After that, add 5 ml of complete medium to resuspend the cells and transfer them to a 25 cm 2 cell culture flask, and change the medium and passage according to the cell growth situation.

[0034] 3. Determination of GiSB growth curve

[0035] After digesting the GiSB cells in a T25 cell culture flask, with an initial concentration of 4×10 4Cells were inoculated into several 12-well plates, and L-15 medium containing 10% fetal bovine serum was added. After adherent growth in an incubator at 25°C for 2 hours, the cells were placed in incubators at 16°C, 25°C, and 30°C for culture respectively. Every day, 3 wells of cells were taken out from each experimental group, digested with trypsin, and the cells were collected. A hemocytometer was used to count the cells. The growth curves of the cells at different culture temperatures were plotted continuously for 7 days. The effects of different culture temperatures on cell growth are as Figure 2 shown. GiSB cells can grow at all three temperatures, and the optimal growth temperature is 25°C. The growth rate at 16°C or 30°C is significantly lower than the optimal growth temperature of 25°C.

[0036] 4. Karyotype analysis of GiSB

[0037] The 20th passage of GiSB cells was transferred to a T25 cell culture flask. After culturing at 25°C for 48 hours, a colchicine solution with a final concentration of 200 μg / mL was added and allowed to act for 6 hours. After digestion with trypsin, the cells were collected by centrifugation at 1000 rpm for 10 minutes and resuspended in a hypotonic solution (0.075 M KCl). They were incubated in a water bath at 37°C for 30 minutes, and the cells were collected again under the above centrifugation conditions. Fresh Carnoy's fixative (methanol: glacial acetic acid = 3:1) was added and fixed at room temperature for 25 minutes, and the fixation step was repeated 2 times. Finally, the cells were resuspended in 300 μl of fresh fixative and gently pipetted to mix evenly. The cell suspension was aspirated and vertically dropped onto a glass slide pre-cooled to -20°C from a height of about 1 m. After drying, it was stained with 10% Giemsa stain for 2 hours, rinsed with pure water to remove the stain, and dried again. At least 100 cells in the metaphase of mitosis were observed under a 100× oil immersion lens, photographed, and the chromosome number was calculated. 100 mitotic figures of GiSB cells were counted respectively. The results are as Figure 3 shown in Figure A. The karyotype of GiSB, a cell line from the swim bladder of Carassius auratus gibelio, is 3n = 156, accounting for 46% of the chromosome numbers of the observed mitotic figures.

[0038] 5. Determination of the sensitivity of GiSB to Carassius auratus herpesvirus

[0039] Virus sensitivity assay was performed using the Carassius auratus herpesvirus (CaHV) preserved in our laboratory. The GiSB cells were passaged into a T25 cell culture flask until 80%-90% of the bottom of the flask was covered, and after adherent culture for 12 h, 500 μl of the CaHV virus suspension was inoculated and adsorbed at 25 °C for 1 hour. After the adsorption was completed, the old culture medium was aspirated, and 5 ml of L-15 medium containing 2% fetal bovine serum was added, and the cells were cultured in an incubator at 25 °C. Photos were taken and observed with an inverted microscope every day. After three blind passages, obvious cytopathic effects (CPE) were still observed in the cells on the third day, including cell shrinkage, rounding, detachment, cell fusion, and vacuolization, etc. The results are shown in Figure 4 Figure A. The GiSB cells infected with CaHV were harvested at 0-5 days post-infection (dpi). RNA of the samples was extracted using the Trizol method, and its cDNA was obtained after reverse transcription. CaHV MCP was amplified by PCR to detect whether the viral gene was expressed. The results are shown in Figure 4 Figure B. In the GiSB cells infected with CaHV, clear bands could be detected 1 day later, and the bands gradually deepened in subsequent detections, indicating that the expression level of CaHV MCP gradually increased with the increase of the number of days of virus infection.

[0040] To further clarify the virus infection, the cells infected with CaHV were gently scraped with a cell scraper, prepared into ultra-thin sections, and observed using a transmission electron microscope. The results are shown in Figure 4 Figure C. The nuclei of the GiSB cells infected with CaHV became larger, and a large number of neatly arranged virus nucleocapsids with a diameter of about 100 nm could be observed in the nuclei, further proving that CaHV can proliferate and replicate in GiSB. The above results indicate that GiSB cells are sensitive to CaHV and can be used as a tool to study the virus infection mechanism and virus-host interaction.

[0041] 6. Sensitivity assay of GiSB to other aquatic viruses

[0042] The GiSB cells were inoculated with Andrias davidianus ranavirus (ADRV), Rana gryliovirus (RGV), Grass carp reovirus (GCRV), Paralichthys olivaceus rhabdovirus (PORV), and Scophthalmus maximus rhabdovirus (SMRV) preserved in our laboratory at an MOI of 0.01 for virus sensitivity assays. Photos were taken and RNA was extracted under a microscope at 12 h, 24 h, and 48 h post-infection, followed by reverse transcription to cDNA, and the virus expression was detected by PCR.

[0043] As Figure 5 shown, the above five aquatic viruses could all cause cytopathic effects in GiSB cells, including cell rounding, shrinkage, and detachment, etc. The PCR results showed that the expression of viral genes could be detected for SMRV, RGV, PORV, and RGV at 12 h post-infection, and for GCRV at 36 h post-infection. Therefore, this cell line could serve as an effective tool for the isolation and detection of various aquatic viruses.

[0044] 7. Expression test of foreign genes in GiSB

[0045] To detect the efficiency of GiSB cells in expressing foreign genes, the plasmid pEGFP-N3 encoding green fluorescent protein was transfected into GiSB cells, and the expression efficiency was detected by observing the fluorescence area. The specific steps were as follows: The GiSB cells were passaged into a 12-well culture plate and cultured in an incubator at 25 °C. When the density of GiSB cells reached 90%, transfection was carried out according to the operating steps of Lipomaster3000 Transfection Reagent (Novoprotein). Take two 1.5 ml EP tubes. Add 50 μl of Opti-MEM serum-free medium and 3 μl of Lipomaster 3000 Reagent to tube A, and add 50 μl of Opti-MEM serum-free medium, 2 μl of T3000 Enhancer Reagent, and 1 μg of pEGFP-N3 plasmid to tube B. Mix A and B and let them stand at room temperature for 20 min. Then add them to the GiSB cells in the 12-well culture plate. After culturing for 12 hours, aspirate the old medium containing the transfection reagent and replace it with fresh L-15 complete medium. Observe and take photos under a fluorescence microscope at 24 h and 48 h post-transfection.

[0046] As Figure 6As shown, 24 hours after transfection, a small number of GiSB cells were observed to emit green fluorescence; after 48 hours, the green fluorescence gradually became stronger and spread throughout the entire field of view. This indicates that the GISB cell line can express foreign genes relatively efficiently. This has important value for the research on the expression, localization, and interaction of foreign genes in GiSB cells.

[0047] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Carassius auratus gibelio swim bladder cell line, characterized in that, Preserved in the China Center for Type Culture Collection on December 10, 2024, with the preservation number: CCTCC NO: C202476.

2. Application of the allogynogenetic gibel carp swim bladder cell line described in claim 1 in protein expression.

3. Application of the allogynogenetic gibel carp swim bladder cell line described in claim 1 in isolating or culturing aquatic animal viruses.

4. Application of the allogynogenetic gibel carp swim bladder cell line described in claim 1 in detecting aquatic animal viruses.

5. A method for detecting aquatic animal viruses, characterized in that, Comprising the following steps: S1: Adding the sample to be detected into the culture environment of the allogynogenetic gibel carp swim bladder cell line described in claim 1; S2: Observing the characteristic changes of the allogynogenetic gibel carp swim bladder cell line cultured in S1.

6. The method according to claim 5, wherein The said characteristic changes are cell morphological changes.

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