Mandarin fish medulla oblongata tissue cell line as well as construction method and application thereof
The tissue cells of the mandarin fish bulbar bulbar tissue were treated by trypsin digestion, and a stable and sensitive mandarin fish bulbar bulbar cell line MFSB was constructed, solving the problem of difficulty in establishing a stable mandarin fish bulbar cell line in the prior art, and achieving a broad-spectrum sensitivity to a variety of fish viruses and an efficient virus research tool.
Patent Information
- Application Number
- CN202510060072.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-04-11
AI Technical Summary
It is difficult to establish a stable medulla of mandarin fish medulla cell line in the prior art, and the sensitivity and broad spectrum of existing cell lines to viruses have not been fully utilized.
The medulla bulbar tissue of the mandarin fish was digested and treated by putting it into trypsin digestion solution. The digested cells were collected and cultured for primary culture and subculture, and the mandarin fish medulla bulbar cell line MFSB was constructed.
The successfully constructed mandarin fish medulla bulbar cell line MFSB has broad-spectrum sensitivity to a variety of fish viruses, is stable in passage, and can be continuously passed to more than 70 generations. It also provides an effective tool for the isolation, culture, detection and vaccine development of fish viruses.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of aquatic biological cells, and in particular to a mandarin fish medulla oblongata cell line MFSB and a construction method and application thereof. Background Art
[0002] Mandarin fish (Sinipercascherzeri) belongs to the order Perciformes, family Serranidae, genus Siniperca. Mandarin fish farming has the characteristics of high economic benefits, tender and smooth meat, no intramuscular spines, and high nutritional value. Mandarin fish has great development potential in terms of market demand, farming technology, ecological benefits, policy support, and supply chain improvement. With the increasing maturity of artificial breeding technology, the scale of farming has continued to expand, and various diseases have become increasingly prominent, posing a certain threat to the mandarin fish farming industry. Among them, viral diseases are the most harmful to the breeding of Siniperca chuatsi seedlings, such as Infection spleen and kidney necrosis virus (ISKNV), Mandarin fish ranavirus (MRV), Siniperca chuatsi rhabdovirus (SCRV) and Siniperca scherzeri birnavirus (SCBV). These viral diseases have different onset seasons, onset cycles and symptoms, but viral diseases break out quickly, causing huge losses to young and adult fish. The virus can infect more fish through horizontal and vertical transmission, causing a large number of fish to become sick, with a mortality rate of more than 90%.
[0003] The frequent outbreak of diseases has restricted the rapid development of mandarin fish farming, among which the most serious is the outbreak of viral diseases. The role of fish cell lines in fish farming has been increasingly valued and has great application value. Fish cell lines can be used for the isolation and identification of viruses, the study of the functions of specific genes, the discovery of specific biomarkers, the screening and toxicity assessment of drugs, and the preparation of vaccines. They have the advantages of low cost, good repeatability, easy access to materials, and precise control of experimental conditions. The establishment of fish cell lines can not only improve the accuracy of experiments, but also help to conduct in-depth research on the physiological and pathological mechanisms of fish. The technology for establishing primary fish cell lines is currently relatively mature, but it is still challenging to obtain a cell line that is sensitive to viruses. In view of the existing problems, the most effective way to establish a stable fish cell line is to use quantity to quality, that is, to prepare a large number of primary cell lines, so as to screen out sensitive cell lines.
[0004] At present, there are two main methods for primary cell culture of fish cells: tissue block method and trypsin digestion method. The tissue block method is mainly used for cell culture when the materials used are small. This method is to cut the tissue block into small pieces, transfer it to a culture bottle, and culture it under a suitable environment. For example, the fin, muscle and heart cells of koi, and the snout cells of yellowfin fish can be cultured by this method. The implementation steps of the trypsin digestion method are to treat the tissue with a trypsin solution, digest it by heating in a water bath, separate the tissue cells, and inoculate the separated cell samples obtained by filtering and other methods into a culture bottle for culture. For example, the skin cells of Chinese gill fish, the epithelial cells of carp, and the liver cells of yellowfin fish can be cultured by this method. However, both methods will cause mechanical damage during the operation. Among the many established fish cell lines, there are no reports on the establishment of the medullary cell line of mandarin fish.
[0005] In order to successfully establish a fish cell line, researchers mainly examine whether the cell line can be stably propagated, its sensitivity to viruses, and the broad-spectrum test of viruses. Whether primary cells can be stably inherited is based on the reliability of data after multiple passaging, and the latter two are based on the feasibility of conducting virus infection experiments. The current method for constructing mandarin fish cell lines still has the following problems: First, in terms of stable passage, many cell lines can be passaged for more than 30 generations, and fewer cell lines can be stably passaged for more than 60 generations. If they are to be passaged for more than 60 generations, optimization adjustments need to be made in the culture medium and serum; secondly, many cell lines have not made too many infection models for virus sensitivity, and many cell lines do not show broad-spectrum virus, only infecting 1-2 viruses, and there is no public TCID for viral infection. 50 In addition, most cell lines are brain cells, spinal cord cells and other cell lines, and no prior art has studied the possibility of using medullary cells in constructing cell lines. Summary of the invention
[0006] In view of the shortcomings of the prior art, the present invention provides a mandarin fish medulla oblongata cell line MFSB with strong division ability, fast growth rate, good stability, low culture cost and broad-spectrum sensitivity to viruses, as well as a construction method and application thereof. The cell line is stable in passage and has a broad spectrum of sensitivity to mandarin fish infectious spleen and kidney necrosis virus, mandarin fish frog iridovirus, mandarin fish rhabdovirus and bi-segmented RNA virus.
[0007] In a first aspect of the present invention, a mandarin fish spinal bulb cell line MFSB is provided, which was deposited in China Center for Type Culture Collection on December 4, 2024, with a deposit number of CCTCC NO: C2024376, and named: Mandarin fish spinal bulb cell line MFSB (Mandarinfish spinal bulb). The depository address is Wuhan University, Wuhan, China.
[0008] Furthermore, the mandarin fish medulla oblongata cell line MFSB is constructed by digesting mandarin fish medulla oblongata tissue in pancreatic enzyme, collecting the digested cells for primary culture and subculture.
[0009] The second aspect of the present invention is a method for constructing the mandarin fish medulla oblongata cell line MFSB as claimed in claim 1 or 2, wherein the method comprises the following steps:
[0010] Step S1, placing the medulla oblongata tissue of mandarin fish in a trypsin digestion solution containing EDTA for digestion, and collecting the digested cells by filtering through a sieve;
[0011] Step S2, adding the collected mandarin fish medullary cells to L-15 cell culture medium containing fetal bovine serum to prepare a cell suspension for primary culture;
[0012] Step S3: When the primary cultured cells have grown all over the bottom of the culture flask, the culture medium is removed, and the cells are digested with trypsin digestion solution. After digestion, the trypsin is discarded, and L-15 cell culture medium containing fetal bovine serum is added to resuspend the cells, and the resuspended cells are subcultured.
[0013] Furthermore, the method for obtaining the medulla oblongata tissue of mandarin fish in step S1 is: taking fresh mandarin fish, dissecting the medulla oblongata tissue of mandarin fish under sterile conditions, soaking it in a penicillin-streptomycin double antibody solution with a concentration of 100u / ml, and immersing it 2 to 3 times.
[0014] Furthermore, the pancreatic digestion solution in step S2 and step S3 contains 0.25% w / v pancreatic enzyme and 0.02% w / v EDTA, the digestion time is 20 to 30 minutes, and the digestion temperature is 37°C.
[0015] Furthermore, the L-15 cell culture medium in step S2 and step S3 contains 20% fetal bovine serum; the primary culture temperature in step S2 is 25°C to 28°C, and more preferably, the primary culture temperature is 28°C.
[0016] Furthermore, after the cells are preliminarily digested with trypsin in step S3, the digested cells are placed in L-15 culture medium for subculture, the subculture ratio is 1:1 to 1:2, and the cells are subcultured once every 2 to 3 days.
[0017] The third aspect of the present invention is the use of the mandarin fish medullary cell line MFSB as claimed in claim 1 or 2 in the isolation, culture, detection and vaccine development of fish viruses.
[0018] Furthermore, the virus is mandarin fish infectious spleen and kidney necrosis virus, mandarin fish bi-segmented RNA virus, mandarin fish frog iridovirus, or mandarin fish rhabdovirus.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The mandarin fish medulla oblongata cell line MFSB of the present invention is successfully cultured in vitro for the first time, and the medulla oblongata tissue cells are separated by short-term digestion with trypsin. After culturing for 2 days, the cells obviously show extended growth; after 4 days, the culture dish is covered with the cells, and after digestion, the cells have strong reproductive ability and present a typical fibroblast morphology; the cells are continuously subcultured for 6 months and have been subcultured to the 50th generation, and the fibroblast morphological characteristics and growth characteristics are still maintained; even after continuous culture for 70 generations, the cells still maintain strong growth characteristics and proliferation characteristics, and a large number of mandarin fish-derived cells can be provided.
[0021] The cell line of the invention has stable characteristics, and has been propagated to 90 generations, and the cells can maintain a good growth state and can be cryopreserved. The mandarin fish medulla oblongata cell line of the invention is sensitive to a variety of fish viruses, and cell pathological changes occur after infection, and can be directly applied to pathogen research. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is a cell morphology diagram of the medullary cell line of mandarin fish under a phase contrast microscope in Example 2 of the present invention.
[0023] Figure 2 This is a growth curve of the mandarin fish medullary cell line under different culture media in Example 3 of the present invention.
[0024] Figure 3 This is a growth curve of the mandarin fish medullary cell line in culture medium with different serum concentrations in Example 3 of the present invention.
[0025] Figure 4 This is a growth curve of the mandarin fish medullary cell line at different culture temperatures in Example 3 of the present invention.
[0026] Figure 5 This is a statistical result diagram of the karyotype analysis of the medullary cell line of mandarin fish in Example 4 of the present invention.
[0027] Figure 6 This is a cell morphology picture of the medullary cell line of mandarin fish under an inverted microscope after virus inoculation in Example 5 of the present invention.
[0028] Figure 7 The TCID of the virus titer in the mandarin fish medullary cell line was calculated using the Reed-Muench method in Example 6 of the present invention. 50 The measurement result diagram. DETAILED DESCRIPTION
[0029] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention. The following is to describe the principles and features of the present invention, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. In the description of the present invention, it should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. Those without specifying the manufacturer for reagents or instruments used are all conventional products that can be obtained by commercial purchase.
[0030] Example 1: Construction method of mandarin fish medullary cell line
[0031] This embodiment is used to provide a method for constructing the mandarin fish medullary cell line of the present invention, which mainly includes the steps of separation, primary culture and subculture, and the specific method is as follows.
[0032] 1. Experimental Materials and Reagents
[0033] 1) Experimental animals: Five mandarin fish weighing 10 g to 15 g were randomly selected (healthy mandarin fish that had been repeatedly identified as not infected with common pathogens and viruses).
[0034] 2) Experimental equipment: dissecting scissors, ophthalmic scissors, ophthalmic tweezers and alcohol cotton, etc.
[0035] 3) Culture medium and related reagents: penicillin-streptomycin double antibody solution with a concentration of 100u / ml; cell culture medium: L-15 culture medium (Gibco) containing 100U / ml penicillin-streptomycin double antibody and 20% fetal bovine serum (Gibco); trypsin digestion solution (0.58g sodium hydrogen phosphate, 8.0g sodium chloride, 0.4g potassium chloride, 1g glucose, 0.2g EDTA, 2.5g trypsin, 1000ml water); PBS buffer (NaCl 8g, Na2HPO41.44g, KH2PO40.24g, KCl 0.2g, water 1000ml).
[0036] 2. Experimental methods
[0037] The healthy mandarin fish were kept in the laboratory for 7 days and tested for no other diseases. Then the isolation, primary culture and subculture experiments were started. The specific steps are as follows.
[0038] 1) Treatment of medullary tissue: Take fresh mandarin fish, dissect the medullary tissue of mandarin fish under sterile conditions, soak it in a penicillin-streptomycin double antibody solution with a concentration of 100u / ml for 10 minutes, and rinse it 2 to 3 times;
[0039] 2) Primary culture: Cut the medulla oblongata tissue into pieces (about 1 mm 3), then digested with 0.25% trypsin digestion solution at 37°C for 20-30 minutes, then added L-15 culture medium to terminate digestion; filtered through a 100μm filter and centrifuged to remove the supernatant, finally added cell culture medium, resuspended and mixed to form a cell suspension; the cell suspension was transferred into culture bottles, cultured in a 25°C-28°C incubator, and the cell adhesion was observed after 2 days of culture. When the cells were plated to 1 / 2, the fresh culture medium was replaced once;
[0040] 3) Subculture: After the cells cover the bottom of the cell bottle (4-5 days), use trypsin digestion solution to preliminarily digest the cells, and then put the digested cells into the culture medium for subculture, with a subculture ratio of 1:1-1:2. In the future, subculture once every 2-3 days according to the growth of the cells.
[0041] Example 2: Cryopreservation of mandarin fish medullary cell line
[0042] This embodiment is used to provide a method for subculture and cryopreservation of the mandarin fish medullary cell line of the present invention, and the specific method is as follows.
[0043] 1. Experimental reagents
[0044] Pancreatic digestion solution: 0.58g sodium hydrogen phosphate, 8.0g sodium chloride, 0.4g potassium chloride, 1g glucose, 0.2g EDTA, 2.5g pancreatin, 1000ml water. Freezing solution: L-15 medium containing 10% DMSO and 40% fetal bovine serum.
[0045] 2. Experimental methods
[0046] When the cells grow to the logarithmic phase, discard the old culture medium and add 1 ml of trypsin digestion solution for digestion; when the cells shrink and become round and fall off the bottle wall, add 2 ml of MFSB cell culture solution to terminate the action of trypsin, gently blow and mix, and count. After centrifugation at 1000 rpm for 5 minutes, remove the culture solution, add cell freezing solution, resuspend the cells, and pack the collected cells into cryopreservation tubes. Indicate the name of the cells, the number of cells and the freezing date on the cryopreservation tubes to obtain the mandarin fish medullary cell line of the present invention, and Figure 1 : is a morphological diagram of the mandarin fish medulla oblongata cell line MFSB of the present invention under a phase contrast microscope, wherein: Figure 1 A is the primary medulla oblongata cell line of mandarin fish; Figure 1 B is the 20th generation mandarin fish medullary cell line; Figure 1 C is the 50th generation mandarin fish medullary cell line; Figure 1 D is the 70th generation mandarin fish medullary cell line. Figure 1 The results showed that the mandarin fish medullary cell line constructed using the construction method of the present invention was still in good condition after 70 generations, indicating that the construction method of the present invention can successfully construct a mandarin fish medullary cell line with stable passage.
[0047] Two months later, the cells were revived according to the conventional cell resuscitation method to verify that the cell proliferation ability was still strong and in good condition. The mandarin fish medullary bulb cell line was deposited in the China Center for Type Culture Collection on December 4, 2024, with the deposit number CCTCC NO: C2024376, and was named: Mandarin fish medullary bulb cell line MFSB (Mandarinfish spinal bulb).
[0048] Example 3: Experimental study on the growth characteristics of mandarin fish medullary cell line MFSB
[0049] This example is used to provide an experimental study on the growth characteristics of the mandarin fish medullary cell line MFSB of the present invention, the purpose of which is to determine the optimal culture medium and the optimal serum concentration of the mandarin fish medullary cell line MFSB. The specific method is as follows.
[0050] 1. Experimental reagents: Pancreatic digestion solution: 0.58g sodium hydrogen phosphate, 8.0g sodium chloride, 0.4g potassium chloride, 1g glucose, 0.2g EDTA, 2.5g pancreatin, 1000ml water; culture medium: L-15, DMEM, MEM, M199 culture medium with different concentrations of fetal bovine serum.
[0051] 2. Experimental methods
[0052] 1) Determination of the optimal culture medium for mandarin fish medullary cell line
[0053] Select four cell culture media: DMEM, L-15, MEM, and M199, and add FBS with a final concentration of 10% to prepare cell culture medium. Adjust the cell density to 2×10 5 cells / mL, and 3 mL / well of each of the four culture media were inoculated into 6-well plates and cultured in a 28°C incubator. Three wells of cells were taken from each experimental group every day, digested with trypsin, collected and counted, and cultured for 7 days. The cells were counted 7 times in succession and their growth curves were drawn. The results are shown in the figure. Figure 2 shown.
[0054] Figure 2 The results showed that the growth rate of the mandarin fish medullary cell line was the highest in the culture medium M199 or L-15 medium, indicating that the optimal culture medium for the mandarin fish medullary cell line is M199 or L-15 medium.
[0055] 2) Determination of the optimal serum concentration for mandarin fish medullary cell line
[0056] Prepare culture medium containing 5%, 10%, 15%, and 20% FBS respectively. Adjust the cell density to 2×10 5cells / mL. Four serum concentrations of culture medium were inoculated into 6-well plates at 3 mL / well and cultured in a 28°C incubator. Three wells of cells were taken from each experimental group every day, digested with trypsin, collected and counted. The cells were cultured for 7 days, counted 7 times in succession, and the growth curve was drawn. The results are shown in Figure 2 shown.
[0057] Figure 3 The results showed that fetal bovine serum (FBS) concentrations of 10% to 20% were suitable for the culture of mandarin fish medullary cells.
[0058] 3) Determination of the optimal culture temperature of mandarin fish medullary cell line
[0059] Four different culture temperatures were selected: 16°C, 24°C, 28°C, and 32°C. L-15 culture medium supplemented with 10% FBS was used and the cell density was adjusted to 2×10 5 cells / mL, the cell suspension was inoculated into 6-well plates at 3 mL / well and placed in four incubators with different culture temperatures. Three wells of cells were taken from each experimental group every day, digested with trypsin, collected and counted, cultured for 7 days, counted 7 times in succession, and the growth curve was drawn. The results are shown in Figure 4 shown.
[0060] Figure 4 The results showed that 28℃ was the most suitable temperature for culturing mandarin fish medullary cells in L-15 medium containing 10% fetal bovine serum.
[0061] Example 4: Karyotype analysis of the mandarin fish medullary cell line MFSB
[0062] This example is used to provide a karyotype analysis experimental study of the mandarin fish medullary cell line MFSB of the present invention, and the specific method is as follows.
[0063] Colchicine at a final concentration of 10 μg / mL was added to the kidney cells of largemouth bass at the 70th generation during the logarithmic growth phase. After treatment at 28°C for 6 hours, the cells were collected and treated with 0.075 mol / L KCl hypotonic solution for 30 minutes. 1 mL of pre-cooled Carnoy's fixative was added, and the cells were centrifuged at 1500 r / min for 5 minutes to remove the supernatant. The cells were fixed with pre-cooled Carnoy's fixative twice, each for 15 minutes. The cold drop method was used for dropping, and after drying, they were stained with 5% Giemsa for 25 minutes. Under microscopic examination, 100 mitotic phases were selected for karyotype analysis and statistics. The results are shown in the figure. Figure 2 shown.
[0064] Figure 5 The results showed that the chromosome mode of the 20th generation mandarin fish medulla oblongata-derived cells was 48, and the chromosome number of the 60th generation mandarin fish medulla oblongata-derived cells was between 27 and 54, with the chromosome mode being 36, 45, and 48.
[0065] Example 5: Experimental study on infection of mandarin fish medullary cell line MFSB with different aquatic viruses
[0066] This example is used to provide an experimental study on the infection of the mandarin fish medullary cell line MFSB of the present invention with different aquatic viruses, and the specific method is as follows.
[0067] A bottle of mandarin fish medullary cells MFSB (P70 generation) with good growth condition was used to adjust the cell density to 2×10 5 cells / mL, the cell suspension was inoculated into a 6-well plate at 3mL / well and placed in a 28℃ incubator. When the cells just covered 90% of the bottom of the bottle, the clean medium was discarded, and the PBS buffer was washed twice. 1mL of diluted fish viruses were inoculated respectively. The fish viruses were: bi-segmented RNA virus (SCBV), infectious spleen and kidney necrosis virus (ISKNV), largemouth bass iris virus (LMBV), and mandarin fish rhabdovirus (SCRV). At the same time, cells treated with PBS were used as blank controls. After 1h of virus incubation, the virus solution was discarded and 2mL of cell maintenance medium (L-15 medium containing 2% FBS) was added. The cell bottle with the virus was placed in a 28℃ incubator. Cell growth and cytopathic effect CPE were observed every day. Once CPE lesions appeared, the cell suspension was collected before the cells were completely detached, and RNA or DNA was extracted, and then PCR or RT-PCR was performed. After blind transmission for three generations, samples were collected for PCR or RT-PCR testing to analyze whether the virus proliferated in the medullary MFSB cells of mandarin fish. The cell lesions after infection were observed under an inverted microscope. Figure 6 shown.
[0068] in, Figure 6 A is the morphological diagram of the cell pathological changes after inoculation with a double-segmented RNA virus (SCBV). Figure 6 B is the morphological picture of the cytopathic effect after inoculation with infectious spleen and kidney necrosis virus (ISKNV); Figure 6 C is the morphological picture of the cell lesions inoculated with largemouth bass iris virus (LMBV); Figure 6 D is the morphological picture of the cell pathological changes after inoculation with Siniperca chuatsi rhabdovirus (SCRV); Figure 6 E is the cell morphology of the blank control (NC) that was not infected with the virus.
[0069] Figure 6 The results of PCR detection showed that after MFSB cells were inoculated with 6 aquatic animal viruses, these 4 viruses showed obvious cytopathic effects in the cells, indicating that the mandarin fish medullary cell line of the present invention can proliferate the viruses SCBV, ISKNV, LMBV, and SCRV, providing a favorable tool for the study of aquatic animal diseases.
[0070] Example 6: TCID of SCBV, ISKNV, LMBV, and SCRV virus-inoculated mandarin fish medullary cell lines 50 Determination experiment
[0071] At present, some studies have been conducted to test the virus sensitivity of ISKNV and SCRV inoculated into the spinal cord tissue of mandarin fish. The Reed-Muench method showed that the titers of ISKNV and SCRV were 10 7.36 , 10 8.7 TCID 50 In order to compare the sensitivity of the mandarin fish medulla oblongata cell line MFSB and mandarin fish spinal cord tissue to ISKNV and SCRV, according to the principle of determining the virus titer by the Reed-Muench method, well-grown MFSB cells were digested and sampled and counted, and the cells were cultured in L-15 medium at a rate of 5×10 4 / mL, and inoculate into 96-well plates with an initial amount of 200μL / well and culture for 24h to allow the cells to fully adhere to the wall. The next day, remove the cell plate and discard the growth medium, add 100μL of L-15 maintenance solution containing 2% FBS to each well, and then add the virus samples to be tested to each well of columns 1 to 10 of the 96-well plate in order of dilution multiples from low to high at 100μL / well, and add 100μL of L-15 maintenance solution containing 2% FBS to each well of columns 11 to 12 as a negative control. The experiment was repeated 3 times. After culturing the cell plate in a 28°C incubator for 96h, observe the cytopathic effect (CPE) and record the endpoint. Calculate the virus titer according to the Reed-Muench method. Set up three parallel experiments, and the results are as follows Figure 7 shown.
[0072] In this embodiment, the calculation formula of virus titer is: virus titer (TCID 50 )=101 g the highest dilution of virus with CPE>50% + (the percentage of viruses with CPE>50% - 50%) / (the percentage of viruses with CPE>50%).
[0073] Figure 7 The results showed that the TCID of ISKNV determined by MFSB 50 The TCID of SCRV is 7.36. 50 The TCID of SCBV is 8.7. 50 The TCID of LMBV is 8.36. 50 is 9.8; it can be seen that ISKNV and SCRV are more sensitive to MFSB cells than mandarin fish spinal cord cells, providing more sensitive and efficient cell tools for subsequent research on virus isolation, culture, detection and vaccine development.
[0074] In summary, the present invention uses trypsin digestion to separate medullary tissue cells from mandarin fish according to the characteristics of the medullary tissue cells of mandarin fish, and performs primary culture, and then successfully constructs a mandarin fish medullary cell line, which has been continuously passaged for more than 70 generations, and can provide a large number of mandarin fish medullary derived cells, and the cells can maintain a good growth state, and can be cryopreserved, providing a good cell model and cell tool for scientific research and animal genetic breeding. In addition, the mandarin fish medullary cell line of the present invention is sensitive to a variety of fish viruses, and obvious cytopathic phenomena occur after infection, and can be applied to the separation, culture, detection and vaccine development of mandarin fish viruses.
[0075] 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 in the protection scope of the present invention.
Claims
1. A mandarin fish medulla oblongata cell line MFSB, characterized in that: It was deposited in the China Center for Type Culture Collection on December 4, 2024, with the deposit number CCTCC NO: C2024376, and named: Mandarin fish medullary tissue cell line MFSB.
2. The mandarin fish medulla oblongata cell line MFSB according to claim 1, characterized in that: The mandarin fish medulla oblongata cell line MFSB is constructed by digesting mandarin fish medulla oblongata tissue in pancreatic enzyme, collecting the digested cells for primary culture and subculture.
3. The method for constructing the mandarin fish medulla oblongata cell line MFSB according to claim 1 or 2, characterized in that: The construction method comprises the following steps: Step S1, placing the medulla oblongata tissue of mandarin fish in a trypsin digestion solution containing EDTA for digestion, and collecting the digested cells by filtering through a sieve; Step S2, adding the collected mandarin fish medullary cells to L-15 cell culture medium containing fetal bovine serum to prepare a cell suspension for primary culture; Step S3: When the primary cultured cells have grown all over the bottom of the culture flask, the culture medium is removed, and the cells are digested with trypsin digestion solution. After digestion, the trypsin is discarded, and L-15 cell culture medium containing fetal bovine serum is added to resuspend the cells, and the resuspended cells are subcultured.
4. The method for constructing the mandarin fish medulla oblongata cell line MFSB according to claim 3, characterized in that: The method for obtaining the medulla oblongata tissue of mandarin fish in step S1 is as follows: taking fresh mandarin fish, dissecting the medulla oblongata tissue of mandarin fish under sterile conditions, soaking it in a penicillin-streptomycin double antibody solution with a concentration of 100u / ml, and washing it 2 to 3 times.
5. The method for constructing the mandarin fish medulla oblongata cell line MFSB according to claim 3, characterized in that: The pancreatic digestion solution in step S2 and step S3 contains 0.25% w / v pancreatic enzyme and 0.02% w / v EDTA, the digestion time is 20 to 30 minutes, and the digestion temperature is 37°C.
6. The construction method according to claim 3, characterized in that: The L-15 cell culture medium in step S2 and step S3 contains 20% fetal bovine serum; the primary culture temperature in step S2 is 25°C to 28°C, and more preferably, the primary culture temperature is 28°C.
7. The construction method according to claim 3, characterized in that: After the cells are preliminarily digested with trypsin in step S3, the digested cells are placed in L-15 culture medium for subculture, with a subculture ratio of 1:1 to 1:2, and subcultured once every 2 to 3 days.
8. Use of the mandarin fish medullary cell line MFSB as claimed in claim 1 or 2 in the isolation, cultivation, detection and vaccine development of fish viruses.
9. The use according to claim 8, characterized in that: The virus is mandarin fish infectious spleen and kidney necrosis virus, mandarin fish bi-segmented RNA virus, mandarin fish frog iridovirus, or mandarin fish rhabdovirus.