Antarctic fish fin cell line as well as construction method and application thereof

Abstract

The invention discloses a Antarctic fish fin cell line and a construction method and application thereof, and the construction method comprises the following steps: step S1: collecting Antarctic fish fin tissues, cleaning and sterilizing, and cutting into pieces to obtain tissue blocks; s2, culturing the tissue blocks in a culture bottle to obtain primary culture cells; s3, when the primary culture cells grow to a certain degree in an adherent manner, trypsin-ED TA is added for digestion, and after the primary culture cells become round and fall off, a complete medium is added, so that the cells continue to grow; s4, when the primary culture cells grow to a certain degree, flushing the cells with PBS, adding trypsin-EDTA digestive juice for digestion, after digestion is completed, resuspending the cells, and carrying out subculture, so that the Antarctic fin cell line is constructed and completed. According to the technical scheme, the Antarctic fish fin cell line is good in growth state, stable in cell proliferation and capable of achieving continuous passage and can be used for expressing exogenous genes for gene function research.

Description

Technical Field

[0001] The invention belongs to marine biological cell culture technology, and specifically relates to a method and application of constructing an Antarctic fish cell line. Background Art

[0002] Antarctic fish usually refers to fish of the suborder Antarctic fish of the order Perciformes that live in the Southern Ocean. They are able to adapt to the extreme environment where the water temperature is maintained below 0°C all year round. During the evolution process, Antarctic fish have undergone a series of changes at the cellular, molecular and genomic levels to adapt to the cold environment of Antarctica. Due to global warming, climate change has caused irreversible impacts on the Antarctic ecosystem, posing a serious threat to the biodiversity and ecological environment of Antarctica.

[0003] Harpagifer antarcticus belongs to the class of ray-finned fish, the order of Perciformes, the family of Gymnospermidae, and the genus of Gymnospermidae. It is distributed in the Southern Ocean, inhabiting depths of 0-84 meters. It can grow up to 9.5 centimeters in length. It is a bottom-dwelling fish, carnivorous, small in size, and its body color is similar to that of the surrounding environment. As an important Antarctic fish, the Harpagifer antarcticus is highly favored in the study of the diversity and adaptive evolution of Antarctic biological resources.

[0004] As an in vitro culture system, fish cell lines are important basic materials for research in virology, toxicology, physiology and molecular genetics. With the rapid development of polar exploration and sample acquisition technology in recent years, research in the field of polar animal life sciences has also made great progress. Uncovering the genome structure of polar animals, especially fishery animals, discovering functional genes, and exploring the molecular mechanisms of polar animals' adaptation to cold environments not only have important scientific significance, but also have great application value and potential.

[0005] However, there is still a gap in the establishment of Antarctic fish cell lines. Therefore, the establishment of Antarctic fish cell lines provides valuable materials for the study of Antarctic fish physiology, toxicology and molecular genetics, and is of great significance for the development of Antarctic fish cell line resources and the preservation of germplasm resources. Summary of the invention

[0006] In view of the defects in the prior art, the purpose of the present invention is to provide an Antarctic fish fin cell line and its construction method and application. The technical solution described in the present invention can obtain a stably propagated Antarctic fish fin cell line, providing important in vitro experimental materials for studying the molecular mechanism of polar fish adaptation to cold.

[0007] In order to achieve the above-mentioned object, the present invention proposes an Antarctic fish fin cell line, and the preservation number of the Antarctic fish fin cell is CCTCC NO: C2024157.

[0008] It should be noted that the Antarctic fish fin cell described in the present invention is the true naked Antarctic fish fin cell HA-Fin-cell2024, classified and named as true naked Antarctic fish (Harpagifer antarcticus), preserved in the China Center for Type Culture Collection (address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province), with a preservation number of CCTCC NO: C2024157, and a preservation date of June 20, 2024.

[0009] In addition, the present invention also proposes a method for constructing the above-mentioned Antarctic fish fin cell line, and the construction method comprises the following steps:

[0010] Step S1: collecting Antarctic fish fin tissue, cleaning and sterilizing it, and cutting it into pieces to obtain tissue blocks;

[0011] Step S2: placing the tissue block into a culture bottle for culture to obtain primary culture cells;

[0012] Step S3: When the primary cultured cells adhere to the wall and grow to 50% to 60% of the bottom of the culture flask, the original culture medium in the culture flask is discarded, the cells are rinsed with PBS (for example, twice), trypsin-EDTA is added for digestion, and after the primary cultured cells become round and fall off, complete cell culture medium is added to allow the cells to be evenly inoculated in the culture flask for continued growth;

[0013] Step S4: When the primary cultured cells grow to 85-95% of the bottom area of ​​the culture medium, the original culture medium in the culture flask is discarded, the cells are rinsed with PBS, and trypsin-EDTA digestion solution is added for digestion. After digestion is completed, complete cell culture medium is added to the culture flask, the cells are collected into a centrifuge tube, centrifuged and the supernatant is discarded, and the cells are resuspended with complete cell culture medium, and the cells are evenly inoculated at a ratio of 1:1 for subculture, thereby completing the construction of the Antarctic fish fin cell line.

[0014] Preferably, the mass volume percentage of the trypsin-EDTA is 0.2-0.3%, preferably 0.25%.

[0015] Preferably, in step S2, the culture temperature is 4°C to 12°C.

[0016] Preferably, the method for preparing the complete cell culture medium is as follows:

[0017] Fetal bovine serum with a volume ratio of 5-25% (preferably 20%), bFGF with a final concentration of 10-15 ng / mL, 2-ME with a final concentration of 50-60 μmol / L, and a penicillin-streptomycin-amphotericin B mixture with a final concentration of 200-250 IU / mL are added to the L-15 basal medium.

[0018] In addition, the present invention provides a method for freezing the above-mentioned Antarctic fish fin cell line, the freezing method comprising the following steps:

[0019] The Antarctic fish fin cells were centrifuged to collect the cell pellets, resuspended in cell freezing solution and stored at -80°C for 24 to 48 hours, and then stored in liquid nitrogen.

[0020] Preferably, the cell freezing solution comprises the following components, each component is expressed in volume percentage:

[0021] 10% DMSO, 10% FBS and 80% L-15 complete medium.

[0022] In some preferred embodiments, the cryopreservation method is specifically performed as follows: the Antarctic fin cell line is grown to a confluence of about 90% and then cryopreserved. The original culture medium in the culture bottle is discarded, the cells are rinsed twice with PBS, and 0.25% trypsin-EDTA digestion solution is added to digest for 3 to 5 minutes. When the cells are basically all rounded and detached from the adherent state, L-15 complete culture medium containing 20% ​​FBS is added to terminate the digestion, and the cells are collected into a centrifuge tube, centrifuged at 1000r / min for 5 minutes, and the cell pellet is collected. The cells are resuspended in a cell freezing solution and stored in a cell freezing tube, and the cell freezing tube is transferred to a programmed cooling box, stored at -80°C for 24 hours, and then transferred to liquid nitrogen for long-term storage. The volume ratio of the components of the cell freezing solution is: 10% DMSO, 10% FBS and 80% L-15 complete culture medium.

[0023] Furthermore, the present invention also proposes a method for resuscitating the above-mentioned Antarctic fish fin cell line, the resuscitation method comprising the following steps:

[0024] The frozen Antarctic fish fin cells were thawed at 12°C to 15°C. When the ice crystals melted, the cells were centrifuged and the supernatant was discarded. Complete cell culture medium was added to resuspend the cells to obtain revived cells.

[0025] Preferably, the resuscitation method further comprises the step of culturing the resuscitated cells in a 10° C., 5% CO 2 incubator.

[0026] In some preferred embodiments, the specific operation of the recovery method is: the cells stored in liquid nitrogen are quickly transferred to a 12°C water bath and gently shaken to thaw. When the ice crystals in the cell cryopreservation tube melt, centrifuge at 1000g for 5 minutes, discard the supernatant, add 3ml of L-15 complete medium to resuspend the cells, and inoculate them into 25cm 2 The culture flask was placed in a 10°C, 5% CO2 incubator for culture. The cell growth status was observed the next day and the culture medium was replaced.

[0027] In addition, the present invention provides one of the following applications of the Antarctic fish fin cell line described above:

[0029] (1) Expression of foreign proteins;

[0030] (2) Preparation of products expressing foreign proteins;

[0031] (3) Construction of cell bank;

[0032] (4) Genetic screening;

[0033] (5) Functional analysis

[0034] In some preferred embodiments, the method for constructing the Antarctic fish fin cell line can adopt the following steps:

[0035] Step S11: After the Antarctic fish is anesthetized, the fin tissue of the Antarctic fish is obtained under sterile conditions. The fin tissue block is cleaned with PBS containing 5% penicillin-streptomycin-amphotericin B, and then the Antarctic fish fin tissue is cut into 1 mm pieces with scissors. 3 Small pieces were washed three times with PBS, and an appropriate amount of complete cell culture medium was added to the tissue pieces;

[0036] Step S12: evenly inoculate the tissue blocks in a culture bottle so that the complete cell culture medium covers the tissue blocks, and culture in a 10° C., 5% CO 2 incubator;

[0037] Step S13: During the first 15 days of the initial culture, the culture medium is replaced every 3 days, and the amount of the replaced culture medium is half of the original culture medium volume;

[0038] Step S14: On the 17th day of culture, when the cells adhere to the wall and grow to 50% to 60% of the bottom of the culture bottle, the original culture medium in the culture bottle is discarded, the cells are rinsed twice with PBS, and trypsin-EDTA digestion solution is added for digestion. After the Antarctic fish fin cells become round and fall off, complete cell culture medium is added to evenly inoculate the cells in the culture bottle.

[0039] Step S15: When the cells grow to 85-95% of the bottom area of ​​the culture medium, the original culture medium in the culture flask is discarded, the cells are rinsed twice with PBS, and trypsin-EDTA digestion solution is added for digestion for 3-5 minutes. During this period, it is best to observe under an inverted microscope that the cells are basically detached from the wall. Otherwise, the digestion time can be appropriately extended. After digestion, 1 mL of complete cell culture medium is added to the culture flask, the cells are collected into a centrifuge tube, centrifuged at 1000 r / min for 5 minutes, the supernatant is discarded, and the cells are resuspended with complete cell culture medium. The cells are evenly inoculated into two culture flasks at a ratio of 1:1, and the culture medium in the flask is supplemented to 3 mL.

[0040] Furthermore, in step S11, the weight of the Antarctic fish is 80±5 g and the body length is 9±2 cm.

[0041] The present invention also provides an identification method for Antarctic fish fin cell lines, wherein the method comprises collecting Antarctic fish fin cells after stable passage, extracting genomes, designing primers using mitochondrial genes, performing PCR amplification, and performing sequence comparison on the sequencing results of PCR amplification using the BLAST tool of NCBI. Combined with the karyotype analysis of cell chromosomes, the source of the Antarctic fish cell line is identified.

[0042] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0043] (1) The existing culture conditions have not established a method for long-term in vitro culture of primary cells of Antarctic fish. The present invention overcomes the gap in the existing technology. On the basis of obtaining primary cells of Antarctic fish, through the exploration and optimization of different culture conditions, a culture medium system and culture conditions suitable for the stable propagation of Antarctic fish fin cells are established, and gene transfection is carried out, which provides technical support for the extensive research of Antarctic fish fin cells.

[0044] (2) The technical solution described in the present invention also provides the identification of Antarctic fish fin cell lines. Through the technical solution described in this case, the Antarctic fish fin cells after stable passage can be collected and the genome can be extracted. The primers are designed using mitochondrial genes, PCR amplification is performed, and the sequencing results of PCR amplification are sequenced using the BLAST tool of NCBI. Combined with the karyotype analysis of the cell chromosomes, the source of the Antarctic fish cell line can be identified.

[0045] (3) The construction method described in the present invention can successfully establish an Antarctic fish fin cell line and can be continuously propagated. It has been propagated for more than 35 generations, thereby providing a large number of Antarctic fish fin cell lines, and the Antarctic fish fin cell line can be directly used in Antarctic fish-related research and functional gene research.

[0046] (4) In the technical solution described in the present invention, the Antarctic fish fin cell line has a good growth state, stable cell proliferation, can be continuously passaged, and can be cryopreserved and revived. The cell line has 48 chromosomes and can be used to express exogenous genes for gene function research. The establishment of this cell line provides valuable in vitro materials for exploring the molecular mechanism of polar animals adapting to cold environments and mining functional genes, and is of great significance to the development of Antarctic fish cell line resources and the preservation of germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The figures show the Antarctic fish fin cells cultured in vitro, wherein (a) is the Antarctic fish fin cells on the 10th day of primary culture, scale bar = 100 μm, and (b) is the Antarctic fish fin cells on the 30th generation, scale bar = 100 μm;

[0048] Figure 2The growth curves of Antarctic fish fin cells are shown, wherein (a) is the growth curve of Antarctic fish fin cell lines in different culture media, and (b) is the growth curve of Antarctic fish fin cell lines at different temperatures;

[0049] Figure 3 The growth curves of the Antarctic fish fin cell line under different FBS contents are shown;

[0050] Figure 4 The PCR amplification and sequence alignment of the mitochondrial gene in the fin cells of Antarctic fish are shown, wherein (a) is the PCR amplification product of the mitochondrial gene in the fin cells of Antarctic fish, and (b) is the alignment analysis of the sequencing sequence of the mitochondrial gene in the cells of Antarctic fish and the mitochondrial gene in Antarctic fish;

[0051] Figure 5 The karyotype analysis of Antarctic fish fin cells is shown, where (a) is the chromosome number distribution of 100 Antarctic fish fin cells at the 22nd generation, (b) is the chromosome of an Antarctic fish fin cell with a diploid number of 48, scale bar = 10 μm, (c) chromosome diploid karyotype, 2n = 48;

[0052] Figure 6 The figure shows the pmaxGFP plasmid transfection of the Antarctic fish fin cell line, where (a) shows the expression of green fluorescent protein (GFP) in the Antarctic fish fin cells, and (b) shows the Antarctic fish fin cells in the bright field. DETAILED DESCRIPTION

[0053] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0054] The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The reagents and raw materials used in the present invention are all commercially available.

[0055] The present invention will be further described below in conjunction with embodiments:

[0056] Example 1. Construction method of Antarctic fish fin cell line

[0057] 1. Preparation of cell culture medium

[0058] The complete cell culture medium used for the Antarctic fin cell line is L-15 basal culture medium supplemented with 20% fetal bovine serum (FBS) (Gibco, A5669701), 10 ng / mL basic fibroblast growth factor (bFGF) (Biyuntian, P5453), 50 μmol / L 2-methoxyestradiol (2-ME) (Sigma, M3184) and 200 IU / mL penicillin-streptomycin-amphotericin B mixture (Solebio, P7630).

[0059] The composition and final concentration of L-15 basal medium are: 900 mg / L D-galactose, 300 mg / L L-glutamine, 550 mg / L sodium pyroglucose, and the pH value is 7.0-7.4.

[0060] 2. Primary Culture

[0061] Under sterile conditions, the fin tissue of a healthy Antarctic fish was placed in 1× phosphate buffered saline (1× PBS) containing 5% penicillin-streptomycin-amphotericin B, and rinsed three times with 1× PBS. The fin tissue was cut into 1 mm thick pieces using sterile scissors. 3 The cleaned fin tissue blocks were then evenly inoculated in a 25 cm 3 Add an appropriate amount of L-15 basal medium to the culture flask, make sure the medium covers the tissue block and does not allow the tissue block to float and move, record the cell information on the side wall of the culture flask, including tissue type, time and culture generation, and place it in a 10°C incubator for static culture. Replace 1mL of fresh L-15 complete medium 24h after cell inoculation, and observe cell migration every day. After observing that cells have migrated out of the tissue block, add 3mL of medium and continue to culture. After that, replace L-15 basal medium every 2 to 3 days.

[0062] 3. Subculture

[0063] When the cells migrate out from the tissue block and grow to 50% to 60% of the bottom area of ​​the culture bottle, the original culture medium in the culture bottle is discarded, the cells are washed twice with 1×PBS, and trypsin is added for digestion. After the Antarctic fish fin cells become round and fall off, the complete cell culture medium is added to make the cells evenly inoculated in the culture bottle. When the cells grow to 85% to 95% of the bottom area of ​​the culture medium, the culture medium is discarded, the cells are washed twice with 1×PBS, and trypsin-EDTA digestion solution is added to digest for 3 to 5 minutes. During this period, pay attention to observation under an inverted microscope. It is appropriate for the cells to basically detach from the wall state. Otherwise, the digestion time can be appropriately extended. After digestion, 1mL of complete cell culture medium is added to the culture bottle, the cells are collected into a centrifuge tube, centrifuged at 1000r / min for 5 minutes, the supernatant is discarded, and the cell is resuspended with complete cell culture medium. It is inoculated into two culture bottles at a ratio of 1:1, and the culture medium in the bottle is supplemented to 3mL. Record cell information on the side wall of the culture bottle, including tissue type, time and culture generation.

[0064] 4. Cell Cryopreservation and Thawing

[0065] 4.1 Cell cryopreservation

[0066] When the Antarctic fin cell line grows to a confluence of about 90%, it is cryopreserved, the original culture medium is discarded, the cells are rinsed twice with PBS, and digested for 3 to 5 minutes with a mass volume percentage of 0.25% trypsin-EDTA digestion solution. When the cells are basically all rounded and separated from the wall state, L-15 basal culture medium containing 20% ​​FBS by volume is added to terminate the digestion, and the cells are collected into a centrifuge tube, centrifuged at 1000r / min for 5 minutes, and the cell pellet is collected. The cells are resuspended in a cell freezing solution and stored in a cell freezing tube, and the cell freezing tube is transferred to a programmed cooling box, stored at -80°C for 24 hours, and then transferred to liquid nitrogen (-196°C) for long-term storage. The volume ratio of each component of the cell freezing solution is: 10% DMSO, 10% FBS and 80% L-15 complete culture medium.

[0067] 4.2 Cell recovery

[0068] When thawing frozen cells, transfer the cells stored in liquid nitrogen to a 12°C water bath and shake gently to thaw. When the ice crystals in the cell cryopreservation tube melt, centrifuge at 1000g for 5 minutes, discard the supernatant, add 3ml of L-15 complete medium to resuspend the cells, and inoculate them into 25cm 2 The culture flask was placed in an incubator at 10°C and a final concentration of 5% CO2 for culture. The cell growth status was observed the next day and the complete cell culture medium was replaced.

[0069] Example 2 Comparison of Growth Conditions of Antarctic Fish Fin Cells

[0070] 2.1 Effects of different culture media on the growth of Antarctic fish fin cells

[0071] The 25th generation Antarctic fish fin cells were cultured at 1×10 5 The cells were seeded at an initial density of 100 cells / well in a 24-well plate. After the cells were stably attached to the wall, the original culture medium was removed and washed twice with 1×PBS. The cells were cultured in L-15 (Gibco, 11415064), MEM (Gibco, C11095500BT), DMEM (Gibco, C11998899BT) and DMEM / F12 (Gibco, C11330500BT) culture media, respectively. 20% FBS, 10 ng / mL bFGF, 50 μmol / L 2-ME and 200 IU / mL penicillin-streptomycin-amphotericin B mixture were added to the above four culture media, and fresh culture media were replaced every 3 days. The cells were cultured in an incubator at 10°C and a final concentration of 5% CO2. The cells in each well of each culture medium were taken on the 1st, 5th, 10th, 15th, 20th and 25th days of culture and counted using a hemocytometer. The experiment was performed in 3 parallels. The growth curve was drawn with the culture time (unit: day) as the horizontal axis and the number of cells as the vertical axis. Figure 2 shown.

[0072] like Figure 2 As shown in (a), it can be seen that L-15 medium is the most suitable medium for the growth of Antarctic fish fin cell lines.

[0073] 2.2 Effects of different culture temperatures on the growth of Antarctic fin cells

[0074] The 26th generation Antarctic fish fin cells were cultured at 1×10 5 The cells were inoculated at an initial density of cells / well in a 24-well plate. After the cells were stably attached to the wall, the original culture medium was removed and rinsed twice with 1×PBS. New L-15 complete culture medium was added and cultured in incubators at 12°C, 10°C and 4°C respectively. The cell culture medium used was L-15 culture medium supplemented with 20% FBS, 10ng / mL bFGF, 50μmol / L2-ME and 200IU / mL penicillin-streptomycin-amphotericin B mixture, and fresh culture medium was replaced every 3 days. The cells were cultured in an incubator at 10°C and a final concentration of 5% CO2, and cells from each well of each culture medium were taken on the 1st, 5th, 10th, 15th, 20th and 25th days of culture and counted with a hemacytometer. The experiment was performed in 3 parallels. A growth curve was drawn with the culture time as the horizontal axis and the number of cells as the vertical axis. The results are as follows Figure 2 shown.

[0075] Depend on Figure 2 (b) shows that 10℃ is the optimal culture temperature for the growth of Antarctic fish fin cell line.

[0076] 2.3 Effects of different fetal bovine serum contents on the growth of Antarctic fish fin cells

[0077] The 27th generation Antarctic fish fin cells were cultured at 1×10 5 The cells were inoculated at an initial density of cells / well in a 24-well plate. After the cells were stably attached to the wall, the original culture medium was removed and the cells were rinsed twice with 1×PBS. The cells were cultured with L-15 basal culture medium with volume fractions of 0%, 5%, 10%, 15%, 20% and 25% FBS, respectively, and fresh culture medium was replaced every 3 days. The cells were cultured in an incubator at 10°C and a final concentration of 5% CO2. Cells from each well of each culture medium were taken on the 1st, 5th, 10th, 15th, 20th and 25th day of culture and counted with a hemocytometer. The experiment was performed in triplicate. A growth curve was drawn with the culture time as the horizontal axis and the number of cells as the vertical axis. The results are shown in Figure 3 shown.

[0078] like Figure 3 As shown, the optimal FBS content for the growth of Antarctic fish fin cell lines is 20%.

[0079] Example 3 Identification of cell origin

[0080] The DNA of the fin cells of the Antarctic fish with good growth at the 30th generation was extracted using a marine animal genomic DNA extraction kit. Specific primers (HA-F: 5'-CTGCCTCTAATCACTAGCTTACT-3', as shown in SEQ ID NO.1, HA-R: 5'-TTCGGATGTCTTGTTCACCTT-3', as shown in SEQ ID NO.2) were designed according to the mitochondrial cell gene of the true naked Antarctic fish (GenBank: MT559889.1) for PCR amplification.

[0081] Among them, MT559889.1 is as follows (see SEQ ID NO.3):

[0082]

[0083] The specific sequence of HAF is as follows (see SEQ ID NO.4):

[0084]

[0085] During amplification, Antarctic fish fin cell DNA was used as a template to prepare a 50 μL PCR reaction system: 25 μL 2×Taq PCR Mix, 2 μL each of upstream and downstream primers, 2 μL DNA template, and 19 μL sterile water.

[0086] PCR reaction conditions: 95℃ pre-denaturation for 5min; 95℃ denaturation for 1min; 58℃ annealing for 1min; 72℃ extension for 1min; 34 cycles, and finally 72℃ extension for 7min. Take 5μL of PCR product for electrophoresis detection in 1% agarose gel and take pictures. After purification, connect the PCR product to the pEASY-T1 vector (full gold, CT111-02) and transform it into Escherichia coli competent cells Trans T1 (full gold, CD501-02). Select positive single clones and send them to the sequencing company for sequencing. The sequencing results are analyzed by sequence comparison using the BLAST tool of NCBI. Results can be found in Figure 3 .Depend on Figure 4 As can be seen from (a), the agarose gel electrophoresis results show that there is a specific band at 1030 bp, which is consistent with the expected target band. The DNA standard (DNA Marker) used is DL2000.

[0087] Depend on Figure 4 As can be seen from (b), after sequencing, BLAST comparison was performed with the sequence in the NCBI database. The results showed that the amplified sequence was 100% consistent with the mitochondrial gene sequence of the true naked Antarctic fish in the NCBI database, indicating that the Antarctic fish cells originated from the true naked Antarctic fish.

[0088] Example 4 Chromosome Analysis

[0089] The 22nd generation of Antarctic fish cells were taken for karyotype analysis. After the cells were cultured in an incubator at 10°C and a final concentration of 5% CO2 for 10 days, colchicine was added to the culture medium to a final concentration of 10μg / ml. After continuing to culture in the incubator for 6 hours, they were digested with trypsin-EDTA digestion solution, centrifuged at 1000g for 5 minutes to collect the cells, the supernatant was discarded, and 5mL of 0.075mol / L KCl solution was added for hypotonicity at 37°C for 30 minutes. Pre-fixed with pre-cooled Carnoy's fixative (methanol: glacial acetic acid = 3:1 ratio, prepared and used immediately) for 2 minutes, centrifuged and discarded the supernatant, and then added Carnoy's fixative for 30 minutes at room temperature, centrifuged at 1000g for 5 minutes, and the supernatant was discarded. Add appropriate amount of Carnoy's fixative according to the amount of cell sediment and blow evenly, use cold drop method to drop the slide at a height of about 30cm, and then use 5% Giemsa stain to stain for 30-40min after drying. Gently rinse off the Giemsa stain with slow water flow, and then observe the chromosomes under a microscope after drying. Select 100 metaphase chromosomes of cell division for photography and statistics. Figure 5 .

[0090] like Figure 5 As shown, Figure 5 (a) shows that the number of chromosomes in the fin cells of Antarctic fish is mainly distributed between 40 and 50. Figure 5 (b) shows that there are 62 cells with 48 chromosomes. Figure 5 (c) shows that a total of 62% of Antarctic fish fin cells have 48 chromosomes (2n=48).

[0091] Example 5 Transfection of GFP reporter gene

[0092] The 22nd generation Antarctic fish cells were digested with trypsin, centrifuged at 1000 g for 5 min to collect the cells, and electroporation working solution (SF 4D-Nucleofector TM X Solution is 16.4μL, Supplement3.6μL, pmaxGFP TM Vector 400ng) to resuspend the cells to a cell number of 2×10 4 Using Lonza electroporator and Amaxa TM 4D-Nucleofector TM Optimization Protocol for Cell Lines electroporation kit was used for electroporation. The cell suspension was added to the electroporation tank of the Lonza electroporator. The electroporation conditions were DS-150. After transfection, 200 μL of complete cell culture medium was added and placed at room temperature for ten minutes. The cells were transferred to a 6-well plate and 3 mL of cell culture medium was added. After transfection for 48 hours, the expression of green fluorescent protein was observed and recorded under a fluorescent inverted microscope. The green fluorescence was observed and photographed. The final results are shown in Figure 6 .

[0093] Figure 6 The figure shows the pmaxGFP plasmid transfection of the Antarctic fish fin cell line, where (a) shows the expression of green fluorescent protein (GFP) in the Antarctic fish fin cells, and (b) shows the Antarctic fish fin cells in the bright field.

[0094] It should be noted that the above-mentioned Antarctic fish fin cell is the true naked Antarctic fish fin cell HA-Fin-cell2024, classified and named as the true naked Antarctic fish (Harpagifer antarcticus), preserved in the China Center for Type Culture Collection (address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province), with the preservation number CCTCC NO: C2024157, and the preservation date is June 20, 2024.

[0095] It should be noted that the prior art in the protection scope of the present invention is not limited to the embodiments given in the present application documents. All prior art that does not contradict the scheme of the present invention, including but not limited to prior patent documents, prior public publications, prior public uses, etc., can be included in the protection scope of the present invention.

[0096] In addition, the combination of the various technical features in this case is not limited to the combination described in the claims of this case or the combination described in the specific embodiments. All technical features recorded in this case can be freely combined or combined in any way unless there is a contradiction between them.

[0097] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. An Antarctic fish fin cell line, It is characterized in that The number of the Antarctic fish fin cell is CCTCC NO: C2024157.

2. A method for constructing the Antarctic fish fin cell line as claimed in claim 1, It is characterized in that The construction method comprises the following steps: Step S1: collecting Antarctic fish fin tissue, cleaning and sterilizing it, and cutting it into pieces to obtain tissue blocks; Step S2: placing the tissue block into a culture bottle for culture to obtain primary cultured cells; Step S3: When the primary cultured cells adhere to the wall and grow to 50% to 60% of the bottom of the culture flask, the original culture medium in the culture flask is discarded, the cells are rinsed with PBS, and trypsin-EDTA is added for digestion. After the primary cultured cells become round and fall off, complete cell culture medium is added to allow the cells to be evenly inoculated in the culture flask and continue to grow; Step S4: When the primary cultured cells grow to 85-95% of the bottom area of ​​the culture medium, the original culture medium in the culture flask is discarded, the cells are rinsed with PBS, and trypsin-EDTA digestion solution is added for digestion. After digestion is completed, complete cell culture medium is added to the culture flask, the cells are collected into a centrifuge tube, centrifuged and the supernatant is discarded, and the cells are resuspended with complete cell culture medium, and the cells are evenly inoculated at a ratio of 1:1 for subculture, thereby completing the construction of the Antarctic fish fin cell line.

3. The construction method according to claim 2, It is characterized in that The mass volume percentage of the trypsin-EDTA is 0.2-0.3%.

4. The construction method according to claim 2, It is characterized in that In the step S2, the culture temperature is 4°C to 12°C.

5. The construction method according to claim 2, It is characterized in that The fish fin tissue is collected from the fin rays of Antarctic fish.

6. The construction method according to claim 2, It is characterized in that The method for preparing the complete cell culture medium is as follows: The L-15 basal medium is supplemented with 5-25% by volume fetal bovine serum, 10-15 ng / mL bFGF at a final concentration, 50-60 μmol / L 2-ME at a final concentration, and 200-250 IU / mL penicillin-streptomycin-amphotericin B mixture at a final concentration.

7. A method for freezing the Antarctic fish fin cell line according to claim 1, It is characterized in that The freezing method comprises the following steps: The Antarctic fish fin cells were centrifuged to collect the cell pellets, resuspended in cell freezing solution and stored at -80°C for 24 to 48 hours, and then stored in liquid nitrogen.

8. The cryopreservation method according to claim 7, It is characterized in that The cell freezing solution includes the following components, each component is calculated by volume percentage: 10% DMSO, 10% FBS and 80% L-15 complete medium.

9. A method for resuscitating the Antarctic fish fin cell line according to claim 1, It is characterized in that The resuscitation method comprises the following steps: The frozen Antarctic fish fin cells were thawed at 12-15°C. When the ice crystals melted, the cells were centrifuged and the supernatant was discarded. Complete cell culture medium was added to resuspend the cells to obtain revived cells.

10. The Antarctic fish fin cell line according to claim 1 is used in one of the following applications: (1) Expression of foreign proteins; (2) Preparation of products expressing foreign proteins; (3) Construction of cell bank; (4) Genetic screening; (5) Functional analysis

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