Golden sand percocypris pingi kidney cell line and construction method thereof

Abstract

The invention provides a percocypris pingi kidney cell line and a construction method thereof, and belongs to the technical field of aquaculture. Wild percocypris pingi is subjected to artificial temporary rearing, kidney tissue is collected, disinfected and crushed into tissue blocks, primary culture and subculture are carried out in an L-15 or DMEM / F12 culture medium containing penicillin, streptomycin, amphotericin B and gentamicin and having the osmotic pressure of 260-300 mOsm / L, and the kidney cell line of the percocypris pingi is obtained. The percocypris pingi kidney cell line obtained by the construction method disclosed by the invention is in a fibroblast shape, can be continuously passed and directly applied to biological characteristic research, and can meet the requirements of germplasm resource preservation and theoretical research of a rare species of the percocypris pingi; and a foundation is laid for research on environmental adaptability of the cell level of the percocypris pingi.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and particularly relates to a kidney cell line of golden sand sea bass and a construction method thereof. Background Art

[0002] Jinsha perch (Percocyprispingi) belongs to the genus Percocypris, family Cyprinidae, order Cypriniformes. It is a rare wild fish unique to my country and is mainly distributed in the upper reaches of the Yangtze River and its tributaries in Sichuan, Guizhou, and Yunnan. Jinsha perch is a national key protected wild animal with a protection level of Class II. In order to protect the germplasm resources of Jinsha perch, various research teams have successively carried out artificial domestication, reproduction, disease prevention and control of Jinsha perch, but research at the cellular level has not been reported.

[0003] Compared with mammalian cell culture, fish cell culture is more prone to contamination, resulting in a higher failure rate, which to a certain extent increases the difficulty of Jinsha sea bass cell culture. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for constructing a Jinsha sea bass kidney cell line, which overcomes the problem of easy contamination during the fish construction process and successfully cultivates the Jinsha sea bass kidney cell line.

[0005] The present invention provides a method for constructing a Jinsha sea bass kidney cell line, comprising the following steps:

[0006] After the wild Jinsha sea bass was temporarily raised artificially, the kidney tissue was collected, disinfected, and minced into tissue blocks. After primary culture and subculture, the Jinsha sea bass kidney cell line was obtained.

[0007] Preferably, during the artificial temporary culture, the temperature of the aquaculture water is 15-20°C, the pH value of the aquaculture water is 7.0-7.5, the dissolved oxygen of the aquaculture water is 6.0-8.0 mg / L, the transparency of the aquaculture water is 70-90 cm, and the potassium permanganate concentration of the aquaculture water is 1-2 mg / L;

[0008] The artificial temporary rearing time is 12 to 24 hours.

[0009] Preferably, the primary culture medium is based on L-15 medium or DMEM / F12 medium, and further comprises 15% to 20% fetal bovine serum, 200 to 300 IU / mL penicillin, 0.2 to 0.3 mg / mL streptomycin, 0.5 to 0.75 μg / mL amphotericin B and 20 to 30 μg / mL gentamicin in a volume percentage concentration;

[0010] The pH value of the primary culture medium is 7.0-7.5, and the osmotic pressure is 260-300 mOsm / L.

[0011] Preferably, when the subculture is subcultured for 1 to 9 generations, the subculture medium is based on L-15 medium or DMEM / F12 medium, and further comprises 100 to 200 IU / mL of penicillin, 0.1 to 0.2 mg / mL of streptomycin, 0.25 to 0.5 μg / mL of amphotericin B, and 10 to 20 μg / mL of gentamicin in a volume percentage concentration; the pH value of the subculture medium is 7.0 to 7.5, and the osmotic pressure is 260 to 300 mOsm / L;

[0012] After the subculture reaches the 10th generation, the cells are cultured with a basic culture medium; the basic culture medium is an L-15 culture medium or a DMEM / F12 culture medium containing 10% to 20% fetal bovine serum by volume; the pH value of the basic culture medium is 7.0 to 7.5, and the osmotic pressure is 260 to 300 mOsm / L.

[0013] Preferably, the temperature of the primary culture or subculture is 15-20°C;

[0014] The primary culture is carried out for 40 to 50 days.

[0015] Preferably, the solvent for kidney tissue disinfection is PBS disinfectant; the PBS disinfectant is a PBS buffer containing 100-300 IU / mL penicillin, 0.1-0.3 mg / mL streptomycin, 0.25-0.75 μg / mL amphotericin B and 10-30 μg / mL gentamicin.

[0016] Preferably, the enzyme solution for digesting primary kidney cells is a 0.1-0.3 g / L EDTA solution containing 0.1%-0.2% trypsin by mass.

[0017] The invention provides a Jinsha sea bass kidney cell line constructed by the construction method.

[0018] The present invention provides a method for preserving the Jinsha sea bass kidney cell line, comprising digesting the Jinsha sea bass kidney cell line in the logarithmic phase into single cells, mixing the cells with a cell freezing solution, and freezing the cells;

[0019] The cell freezing medium is L-15 culture medium or DMEM / F12 culture medium containing 58% to 62% fetal bovine serum and 8% to 12% DMSO.

[0020] The present invention provides a method for resuscitating the Jinsha sea bass kidney cell line, which comprises thawing the frozen Jinsha sea bass kidney cell line in a water bath at 28-32° C., adding a basic culture medium, separating the cells, and resuspending and culturing them in a basic culture medium;

[0021] The basic culture medium is an L-15 culture medium or a DMEM / F12 culture medium containing 10% to 20% fetal bovine serum by volume; the pH value is 7.0 to 7.5, and the osmotic pressure is 260 to 300 mOsm / L.

[0022] The present invention provides a method for constructing a Jinsha perch kidney cell line, comprising the following steps: artificially raising wild Jinsha perch temporarily, collecting kidney tissue, disinfecting it, and crushing it into tissue blocks, and then performing primary culture and subculture to obtain a Jinsha perch kidney cell line. Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The construction method of the present invention is simple and easy to operate, and the construction method is reproducible, and the constructed cell line can be stably propagated;

[0024] 2. Compared with other cell lines, the tissue block contamination rate of the kidney cell line obtained by the construction method of the present invention in the primary culture is low or even no contamination occurs, which greatly improves the success rate of cell line construction;

[0025] 3. The Jinsha sea bass kidney cell line constructed by the present invention has a fibroblast-like morphology, can be continuously passaged and directly applied to the study of biological characteristics, meeting the needs of the preservation of Jinsha sea bass germplasm resources and theoretical research and application;

[0026] 4. Cell culture needs to be combined with the biological characteristics of different species and their complex environmental conditions in the wild. It is not an independent event. The first cell line of Jinsha sea bass was obtained using the construction method of the present invention, which provides a good case for the immediate preservation of germplasm resources of endangered protected animals from the wild. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the morphology of the monolayer of kidney cells of Jinsha sea bass;

[0028] Figure 2 This is a graph showing the effect of culture medium on the growth of kidney cells of Jinsha sea bass. DETAILED DESCRIPTION

[0029] The present invention provides a method for constructing a Jinsha sea bass kidney cell line, comprising the following steps:

[0030] After the wild Jinsha sea bass was temporarily raised artificially, the kidney tissue was collected, disinfected, and minced into tissue blocks. After primary culture and subculture, the Jinsha sea bass kidney cell line was obtained.

[0031] In the present invention, during the artificial temporary culture, the temperature of the aquaculture water body is preferably 15-20°C, and can be 16°C, 17°C, 18°C, or 19°C. The pH value of the aquaculture water body is preferably 7.0-7.5, and can be 7.2 or 7.4. The dissolved oxygen of the aquaculture water body is preferably 6.0-8.0 mg / L, and can be 7.0 mg / L. The transparency of the aquaculture water body is preferably 70-90 cm, and can be 75 cm, 80 cm, or 85. The potassium permanganate concentration of the aquaculture water body is preferably 1-2 mg / L, and can be 1.5 mg / L. The time of the artificial temporary culture is preferably 12-24 h, and can be 14 h, 16 h, 18 h, 20 h, or 22 h. The step of artificial temporary culture is beneficial to reducing the problem of tissue block contamination during the construction of the renal cell line and improving the success rate of the construction of the renal cell line.

[0032] In the present invention, before collecting kidney tissue, the Jinsha sea bass is preferably disinfected, the fish body is anesthetized after being soaked in disinfectant, and then the fish body surface is disinfected. The disinfectant is preferably a 20 mg / L methylene blue solution. The disinfection time is preferably 15 to 20 minutes. The solvent for disinfecting the fish body surface is preferably 75% alcohol.

[0033] In the present invention, the solvent for kidney tissue disinfection is preferably PBS disinfectant; the PBS disinfectant is preferably a PBS buffer containing 100-300 IU / mL penicillin, 0.1-0.3 mg / mL streptomycin, 0.25-0.75 μg / mL amphotericin B and 10-30 μg / mL gentamicin.

[0034] In the present invention, the primary culture medium for primary culture is preferably based on L-15 medium or DMEM / F12 medium, and further comprises 15% to 20% fetal bovine serum, 200 to 300 IU / mL penicillin, 0.2 to 0.3 mg / mL streptomycin, 0.5 to 0.75 μg / mL amphotericin B and 20 to 30 μg / mL gentamicin in volume percentage concentration; it can be 18% fetal bovine serum, 250 IU / mL penicillin, 0.25 mg / mL streptomycin, 0.65 μg / mL amphotericin B and 25 μg / mL gentamicin in volume percentage concentration. The pH value of the primary culture medium is 7.0 to 7.5, and can be 7.2 or 7.4; the osmotic pressure is 260 to 300 mOsm / L, and can be 280 mOsm / L. The temperature of the primary culture is 15-20°C, and can be 16°C, 17°C, 18°C, or 19°C. The primary culture is inoculated in a cell culture bottle, inverted and cultured overnight, and the primary culture medium is replaced every 3.5 days. The number of days of the primary culture is preferably 40-50 days, and can be 45 days. When the primary culture is carried out for 25 days, the cells migrate from the tissue block and grow into a cell monolayer when cultured for 45 days.

[0035] In the present invention, the enzyme solution for digesting primary kidney cells is a 0.1-0.3 g / L EDTA solution containing 0.1%-0.2% trypsin by mass percentage, and can be a 0.2 g / L EDTA solution containing 0.15% trypsin by mass percentage.

[0036] In the present invention, when the subculture is subcultured for 1 to 9 generations, the subculture medium is based on L-15 medium or DMEM / F12 medium, and further comprises 100 to 200 IU / mL of penicillin, 0.1 to 0.2 mg / mL of streptomycin, 0.25 to 0.5 μg / mL of amphotericin B and 10 to 20 μg / mL of gentamicin in a volume percentage concentration; the pH value of the subculture medium is 7.0 to 7.5, and the osmotic pressure is 26 0~300mOsm / L; it can be 120~180IU / mL of penicillin, 0.12~0.18mg / mL of streptomycin, 0.3~0.4μg / mL of amphotericin B and 12~18μg / mL of gentamicin in volume percentage concentration; it can also be 150IU / mL of penicillin, 0.15mg / mL of streptomycin, 0.35μg / mL of amphotericin B and 15μg / mL of gentamicin in volume percentage concentration. The pH value of the subculture medium is preferably 7.0~7.5, and can be 7.2 and 7.4. The osmotic pressure is preferably 260~300mOsm / L, and can be 270mOsm / L, 280mOsm / L, 290mOsm / L. After the subculture reaches the 10th generation, the cells are cultured with a basal medium; the basal medium is an L-15 medium or a DMEM / F12 medium containing 10% to 20% fetal bovine serum by volume; the concentration of the fetal bovine serum can be 12% to 18%, more preferably 15%. The pH value of the basal medium is 7.0 to 7.5, and the osmotic pressure is 260 to 300 mOsm / L. The temperature of the subculture is preferably 15 to 20°C, can be 16 to 18°C, or can be 17°C.

[0037] In the embodiments of the present invention, by optimizing the types of culture medium used in primary culture and subculture, the results show that DMEM, DMEM / F12, L-15, M199, and M1640 are used as basal culture medium. DMEM / F12 and L-15 culture medium have the fastest cell growth rate and the most cell adherence growth, which are most suitable for the culture of kidney cells of golden sand sea bass.

[0038] The present invention provides a Jinsha perch kidney cell line constructed by the construction method. The Jinsha perch kidney cell line is fibroblastic. The Jinsha perch kidney cell line is the first cell line of Jinsha perch, which fills the gap of current cell materials and lays a foundation for the study of environmental adaptability of Jinsha perch cell level.

[0039] The invention provides a method for preserving the Jinsha sea bass kidney cell line. The Jinsha sea bass kidney cell line in the logarithmic phase is digested into single cells and then mixed with a cell freezing solution for freezing. The cell freezing solution is an L-15 culture medium or a DMEM / F12 culture medium containing 58% to 62% fetal bovine serum and 8% to 12% DMSO.

[0040] The digestion method of the present invention is the same as the digestion method in the above technical solution, which will not be described in detail here. The concentration of the digested Jinsha carp kidney cell line single cell in the cell freezing solution is preferably 0.5×10 6 / mL~10×10 6 / mL, which can be 1×10 6 The cell freezing solution is L-15 culture medium or DMEM / F12 culture medium containing 60% fetal bovine serum and 10% DMSO. The freezing method preferably places the cell freezing solution in a program cooling box at -80°C for 48 hours, and then places it in liquid nitrogen for long-term storage.

[0041] The invention provides a method for resuscitating the Jinsha sea bass kidney cell line. The method comprises the following steps: thawing the frozen Jinsha sea bass kidney cell line in a water bath at 28-32 DEG C, separating the cells after adding a basic culture medium, and resuspending and culturing the cells in a basic culture medium. The basic culture medium is an L-15 culture medium or a DMEM / F12 culture medium containing fetal bovine serum with a volume percentage concentration of 10%-20%, a pH value of 7.0-7.5, and an osmotic pressure of 260-300mOsm / L; or an L-15 culture medium or a DMEM / F12 culture medium containing fetal bovine serum with a volume percentage concentration of 15%, a pH value of 7.3, and an osmotic pressure of 280mOsm / L.

[0042] The following is a detailed description of a Jinsha sea bass kidney cell line and a construction method thereof provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] A method for constructing a Jinsha sea bass kidney cell line

[0045] 1. Preparation of PBS disinfectant and cell culture medium

[0046] PBS disinfectant: Add antibiotics to PBS to a concentration of 300 IU / mL penicillin, 0.3 mg / mL streptomycin, 0.75 μg / mL amphotericin B, and 30 μg / mL gentamicin.

[0047] Primary culture medium: Add fetal bovine serum and antibiotics to L-15 culture medium so that fetal bovine serum accounts for 20% of the total volume, the concentration of penicillin is 200 IU / mL, the concentration of streptomycin is 0.2 mg / mL, the concentration of amphotericin B is 0.5 μg / mL, and the concentration of gentamicin is 25 μg / mL.

[0048] Subculture medium: Add fetal bovine serum and antibiotics to L-15 culture medium so that the fetal bovine serum accounts for 15% of the total volume, the concentration of penicillin is 100 IU / mL, the concentration of streptomycin is 0.1 mg / mL, the concentration of amphotericin B is 0.3 μg / mL, and the concentration of gentamicin is 15 μg / mL.

[0049] Basal culture medium: Add fetal bovine serum to L-15 culture medium so that the fetal bovine serum accounts for 15% of the total volume.

[0050] The pH value of the culture medium was adjusted to 7.0 and the osmotic pressure to 280 mOsm / L, and the culture medium was stored in a 4°C refrigerator for later use.

[0051] 2. Temporary breeding of Jinsha perch and carp

[0052] Jinsha perch were collected from the dam of a power station on the Jinsha River, brought back to the laboratory and temporarily kept overnight. The water temperature was 18-20℃, the pH value was 7.2, the dissolved oxygen was 7.0mg / L, the water transparency was 80cm, and the potassium permanganate concentration was 2mg / L.

[0053] 3. Primary Culture

[0054] The Jinsha perch carp that had been temporarily kept overnight were first disinfected by soaking in 15mg / L methylene blue for 20 minutes, and then anesthetized with 100ppm MS-222; the fish body was then wiped with 75% alcohol, placed in a clean bench, and the kidneys were removed with sterile dissecting instruments, placed in a culture dish, and PBS disinfectant was added to clean the kidney tissue. Washed 4 times, cut into small pieces, and inoculated in a 25cm 2 In the cell culture flask, invert it in a 20℃ incubator overnight, add 5mL of primary culture medium the next day, start primary culture in a 20℃ incubator, replace half of the culture medium every three days, cells begin to migrate from the tissue block on the 20th day, and grow into a cell monolayer on the 40th day ( Figure 1 ).

[0055] 4. Subculture

[0056] After the primary kidney cells covered 80% of the bottom of the bottle, they began to be subcultured. First, the primary culture medium in the culture bottle was aspirated, and 1 mL of 0.2% trypsin-EDTA solution was used for digestion for 2 minutes. After the cells became round and suspended, 2 mL of subculture medium was added to neutralize the trypsin reaction. The bottom of the bottle was gently blown to make the adherent cells fall off. The first three subcultures were subcultured at a 1:1 ratio, and then at a 1:2 ratio. The cells were continued to be cultured in a 20°C incubator; the cells were subcultured every 7 days. At the 10th generation, the cell culture medium was replaced with a basal culture medium. At this time, the Jinsha carp kidney cell line was successfully established.

[0057] 5. Steps for cell freezing and thawing:

[0058] 1) Prepare cell freezing solution: The freezing solution includes L-15 culture medium, fetal bovine serum and DMSO, which are mixed in a volume ratio of 3:6:1 and prepared before use;

[0059] 2) Cell freezing: Take cells in the logarithmic growth phase, digest them with trypsin as described above, centrifuge the cell suspension at 1000 rpm for 6 min, and discard the supernatant; add the prepared cell freezing solution to the cell pellet and resuspend it to a cell concentration of about 1×10 6 / mL, transfer 1mL of cell suspension into a 1.8mL cryovial, place the cryovial in a programmed cooling box at -80℃ for 48h, and then store in liquid nitrogen for long-term storage;

[0060] 3) Cell recovery: Take out the above cryopreserved tubes from liquid nitrogen, put them in a 30℃ water bath and shake them quickly until they melt; then transfer the thawed cells to a 15mL centrifuge tube in a sterile operating table, add an equal amount of basal culture medium, centrifuge at 1000rpm for 6min, and remove the supernatant; resuspend the cells with basal culture medium and transfer them to a cell culture bottle, culture them in a 20℃ incubator, and the cells will grow into a monolayer in 10 days. After recovery, the cell adhesion rate is 85%, and it can be subcultured.

[0061] Example 2

[0062] A method for constructing a Jinsha sea bass kidney cell line

[0063] 1. Preparation of PBS disinfectant and cell culture medium

[0064] PBS disinfectant: Add antibiotics to PBS to a concentration of 200 IU / mL penicillin, 0.2 mg / mL streptomycin, 0.75 μg / mL amphotericin B, and 30 μg / mL gentamicin.

[0065] Primary culture medium: Add fetal bovine serum and antibiotics to L-15 culture medium so that the fetal bovine serum accounts for 15% of the total volume, the concentration of penicillin is 150 IU / mL, the concentration of streptomycin is 0.1 mg / mL, the concentration of amphotericin B is 0.6 μg / mL, and the concentration of gentamicin is 20 μg / mL.

[0066] Subculture medium: Add fetal bovine serum and antibiotics to L-15 culture medium so that the fetal bovine serum accounts for 15% of the total volume, the concentration of penicillin is 100 IU / mL, the concentration of streptomycin is 0.1 mg / mL, the concentration of amphotericin B is 0.4 μg / mL, and the concentration of gentamicin is 15 μg / mL.

[0067] Basal culture medium: Add fetal bovine serum to L-15 culture medium so that the fetal bovine serum accounts for 10% of the total volume.

[0068] The pH value of the culture medium was adjusted to 7.2 and the osmotic pressure to 300 mOsm / L, and the culture medium was stored in a 4°C refrigerator for later use.

[0069] 2. Temporary breeding of Jinsha perch and carp

[0070] Jinsha perch were collected from the dam of a power station on the Jinsha River, brought back to the laboratory and temporarily kept overnight. The breeding water temperature was 15-20℃, the pH value of the water was 7.0, the dissolved oxygen of the water was 8.0mg / L, the water transparency was 80cm, and the potassium permanganate concentration was 1.5mg / L.

[0071] 3. Primary Culture

[0072] The Jinsha perch carp that was temporarily kept overnight was first disinfected by soaking in 20mg / L methylene blue for 30 minutes, and then anesthetized with 100ppm MS-222; the fish body was then wiped with 75% alcohol and placed in a clean bench, and the kidney was removed with a sterile dissecting instrument and placed in a culture dish. PBS disinfectant was added to clean the kidney tissue. After washing 5 times, it was cut into small pieces and inoculated in a 25cm 2 In the cell culture flask, invert it in an 18°C ​​incubator overnight. The next day, add 5 mL of primary culture medium and start primary culture in an 18°C ​​incubator. Replace half the amount of culture medium every three days. On the 30th day, cells begin to migrate from the tissue block and grow into a cell monolayer on the 50th day.

[0073] 4. Subculture

[0074] After the primary kidney cells covered 80% of the bottom of the bottle, they began to be subcultured. First, the primary culture medium in the culture bottle was aspirated, and 1 mL of 0.1% trypsin-EDTA solution was used for digestion for 3 minutes. After the cells became round and suspended, 2 mL of subculture medium was added to neutralize the trypsin reaction. The bottom of the bottle was gently blown to make the adherent cells fall off. The first three subcultures were subcultured at a 1:1 ratio, and then at a 1:2 ratio. The cells were continued to be cultured in an incubator at 18°C; the cells were subcultured every 7 days. At the 10th generation, the cell culture medium was replaced with a basal culture medium. At this time, the Jinsha carp kidney cell line was successfully established.

[0075] 5. Steps for cell freezing and thawing:

[0076] 1) Prepare cell freezing solution: The freezing solution includes L-15 culture medium, fetal bovine serum and DMSO, which are mixed in a volume ratio of 3:6:1 and prepared before use.

[0077] 2) Cell freezing: Take cells in the logarithmic growth phase, digest them with trypsin as described above, centrifuge the cell suspension at 1000 rpm for 6 min, and discard the supernatant; add the prepared cell freezing solution to the cell pellet and resuspend it to a cell concentration of about 1×10 6 / mL, transfer 1mL of cell suspension into a 1.8mL cryovial, place the cryovial in a programmed cooling box at -80℃ for 48h, and then store in liquid nitrogen for long-term storage.

[0078] 3) Cell recovery: Take out the above cryopreserved tubes from liquid nitrogen, put them into a 30℃ water bath and shake them quickly until they melt; then transfer the thawed cells to a 15mL centrifuge tube in a sterile operating table, add an equal amount of basal culture medium, centrifuge at 1000rpm for 6min, and remove the supernatant; resuspend the cells with basal culture medium, transfer them to a cell culture bottle, and culture them in an 18℃ incubator. The cells will grow into a monolayer in 10 days. After recovery, the cell adhesion rate is 82%, and it can be subcultured.

[0079] Example 3

[0080] A method for constructing a Jinsha sea bass kidney cell line

[0081] 1. Preparation of PBS disinfectant and cell culture medium

[0082] PBS disinfectant: Add antibiotics to PBS to a concentration of 300 IU / mL penicillin, 0.2 mg / mL streptomycin, 0.5 μg / mL amphotericin B, and 30 μg / mL gentamicin.

[0083] Primary culture medium: Add fetal bovine serum and antibiotics to L-15 culture medium so that the fetal bovine serum accounts for 20% of the total volume, the concentration of penicillin is 200 IU / mL, the concentration of streptomycin is 0.15 mg / mL, the concentration of amphotericin B is 0.5 μg / mL, and the concentration of gentamicin is 25 μg / mL.

[0084] Subculture medium: Add fetal bovine serum and antibiotics to L-15 culture medium so that the fetal bovine serum accounts for 15% of the total volume, the concentration of penicillin is 100 IU / mL, the concentration of streptomycin is 0.1 mg / mL, the concentration of amphotericin B is 0.3 μg / mL, and the concentration of gentamicin is 20 μg / mL.

[0085] Basal culture medium: Add fetal bovine serum to L-15 culture medium so that the fetal bovine serum accounts for 15% of the total volume.

[0086] The pH value of the culture medium was adjusted to 7.0 and the osmotic pressure to 280 mOsm / L, and the culture medium was stored in a 4°C refrigerator for later use.

[0087] 2. Temporary breeding of Jinsha perch and carp

[0088] The Jinsha sea bass came from the rare fish conservation research base. After being brought back to the laboratory, they were temporarily kept overnight. The breeding water temperature was 20℃, the pH value of the water was 7.2, the dissolved oxygen of the water was 7.0mg / L, the water transparency was 80cm, and the potassium permanganate concentration was 2mg / L.

[0089] 3. Primary Culture

[0090] The Jinsha perch carp that was temporarily kept overnight was first disinfected by soaking in 20mg / L methylene blue for 20min, and then anesthetized with 100ppm MS-222; the fish body was then wiped with 75% alcohol and placed in a clean bench, and the kidneys were removed with sterile dissecting instruments, placed in a culture dish, and PBS disinfectant was added to clean the kidney tissue. Wash 4 times, cut into small tissue pieces, and inoculated into a 25cm2 cell culture flask, inverted in a 20℃ incubator overnight, and 5mL of primary culture medium was added the next day, and primary culture was started in a 20℃ incubator. The culture medium was replaced by half every three days. On the 25th day, cells began to migrate from the tissue pieces and grew into a cell monolayer on the 45th day ( Figure 1 ).

[0091] 4. Subculture

[0092] After the primary kidney cells covered 80% of the bottom of the bottle, they began to be subcultured. First, the primary culture medium in the culture bottle was aspirated, and 1 mL of 0.2% trypsin-EDTA solution was used for digestion for 2 minutes. After the cells became round and suspended, 2 mL of subculture medium was added to neutralize the trypsin reaction. The bottom of the bottle was gently blown to make the adherent cells fall off. The first three subcultures were subcultured at a 1:1 ratio, and then at a 1:2 ratio. The cells were continued to be cultured in a 20°C incubator; the cells were subcultured every 7 days. At the 10th generation, the cell culture medium was replaced with a basal culture medium. At this time, the Jinsha carp kidney cell line was successfully established.

[0093] 5. Steps for cell freezing and thawing:

[0094] 1) Prepare cell freezing solution: The freezing solution includes L-15 culture medium, fetal bovine serum and DMSO, which are mixed in a volume ratio of 3:6:1 and prepared before use.

[0095] 2) Cell freezing: Take cells in the logarithmic growth phase, digest them with trypsin as described above, centrifuge the cell suspension at 1000 rpm for 6 min, and discard the supernatant; add the prepared cell freezing solution to the cell pellet and resuspend it to a cell concentration of about 1×10 6 / mL, transfer 1mL of cell suspension into a 1.8mL cryovial, place the cryovial in a programmed cooling box at -80℃ for 48h, and then store in liquid nitrogen for long-term storage.

[0096] 3) Cell recovery: Take out the above cryopreserved tubes from liquid nitrogen, put them in a 30℃ water bath and shake them quickly until they melt; then transfer the thawed cells to a 15mL centrifuge tube in a sterile operating table, add an equal amount of basal culture medium, centrifuge at 1000rpm for 6min, and remove the supernatant; resuspend the cells with basal culture medium, transfer them to a cell culture bottle, and culture them in a 20℃ incubator. The cells will grow into a monolayer in 10 days. After recovery, the cell adhesion rate is 83.4%, and it can be subcultured.

[0097] Comparative Example 1

[0098] A method for constructing a Jinsha sea bass kidney cell line

[0099] The Jinsha sea bass kidney cell line was constructed according to the method in Example 1, except that the temporary culture of the Jinsha sea bass was omitted, and the kidneys of the wild Jinsha sea bass were directly obtained by disinfection and anesthesia.

[0100] The kidney tissue was cut into small pieces and inoculated into 25 cm 2 The cell culture flask was inverted in a 20°C incubator overnight, and the primary culture medium was added the next day to start the primary culture in a 20°C incubator. On the third day, the tissue block was contaminated, the culture medium was turbid, and the primary culture failed.

[0101] Example 4

[0102] A method for constructing a Jinsha sea bass kidney cell line

[0103] The experiment was carried out according to the method described in Example 1, except that the L-15 culture medium in the basal culture medium was replaced with DMEM, DMEM / F12, M199, and M1640, respectively, and different culture mediums were prepared for subculture.

[0104] Results Figure 2 . The results showed that under different culture medium conditions, the cell growth rate and adhesion rate were different. After one day of cell culture, DMEM / F12 and L-15 had more adherent cells, followed by DMEM and M199, while M1640 culture medium had the least adherent cells. With the increase of culture time, the number of cells increased to varying degrees under various culture medium conditions. Among them, the growth rate of cells in DMEM / F12 and L-15 culture medium was the fastest, followed by DMEM and M199, while the growth rate of M1640 cells was the slowest, and the number of cells did not increase significantly. The number of cells in DMEM / F12 and L-15 culture medium was about twice the number of M1640 cells. Therefore, for Jinsha sea bass kidney cells, DMEM / F12 and L-15 have the best effects.

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a Jinsha sea bass kidney cell line, characterized in that: The following steps are involved: After the wild Jinsha sea bass was temporarily raised artificially, the kidney tissue was collected, disinfected, and minced into tissue blocks. After primary culture and subculture, the Jinsha sea bass kidney cell line was obtained.

2. The construction method according to claim 1, characterized in that: During the artificial temporary culture, the temperature of the aquaculture water is 15-20°C, the pH value of the aquaculture water is 7.0-7.5, the dissolved oxygen of the aquaculture water is 6.0-8.0 mg / L, the transparency of the aquaculture water is 70-90 cm, and the potassium permanganate concentration of the aquaculture water is 1-2 mg / L; The artificial temporary rearing time is 12 to 24 hours.

3. The construction method according to claim 1, characterized in that: The primary culture medium is based on L-15 medium or DMEM / F12 medium, and further comprises 15% to 20% fetal bovine serum, 200 to 300 IU / mL penicillin, 0.2 to 0.3 mg / mL streptomycin, 0.5 to 0.75 μg / mL amphotericin B, and 20 to 30 μg / mL gentamicin in a volume percentage concentration; The pH value of the primary culture medium is 7.0-7.5, and the osmotic pressure is 260-300 mOsm / L.

4. The construction method according to claim 1, characterized in that: When the subculture is subcultured for 1 to 9 generations, the subculture medium is based on L-15 medium or DMEM / F12 medium, and further contains 100 to 200 IU / mL of penicillin, 0.1 to 0.2 mg / mL of streptomycin, 0.25 to 0.5 μg / mL of amphotericin B, and 10 to 20 μg / mL of gentamicin in a volume percentage concentration; the pH value of the subculture medium is 7.0 to 7.5, and the osmotic pressure is 260 to 300 mOsm / L; After the subculture reaches the 10th generation, the cells are cultured with a basic culture medium; the basic culture medium is an L-15 culture medium or a DMEM / F12 culture medium containing 10% to 20% fetal bovine serum by volume; the pH value of the basic culture medium is 7.0 to 7.5, and the osmotic pressure is 260 to 300 mOsm / L.

5. The construction method according to claim 1, characterized in that: The temperature of the primary culture or subculture is 15-20°C; The primary culture is carried out for 40 to 50 days.

6. The construction method according to claim 1, characterized in that: The solvent for kidney tissue disinfection is PBS disinfectant; the PBS disinfectant is a PBS buffer solution containing 100-300 IU / mL penicillin, 0.1-0.3 mg / mL streptomycin, 0.25-0.75 μg / mL amphotericin B and 10-30 μg / mL gentamicin.

7. The construction method according to claim 1, characterized in that: The enzyme solution for digesting primary kidney cells is a 0.1-0.3 g / LEDTA solution containing 0.1%-0.2% trypsin by mass percentage.

8. The Jinsha sea bass kidney cell line constructed by the construction method according to any one of claims 1 to 7.

9. The method for preserving the Jinsha sea bass kidney cell line according to claim 8, characterized in that: The logarithmic phase Jinsha sea bass kidney cell line was digested into single cells and then mixed with cell freezing solution and frozen; The cell freezing medium is L-15 culture medium or DMEM / F12 culture medium containing 58% to 62% fetal bovine serum and 8% to 12% DMSO.

10. The method for resuscitating the Jinsha sea bass kidney cell line according to claim 8, characterized in that: The frozen Jinsha carp kidney cell line was thawed in a water bath at 28-32°C, the cells were separated after adding the basic culture medium, and the cells were resuspended and cultured in the basic culture medium; The basic culture medium is an L-15 culture medium or a DMEM / F12 culture medium containing 10% to 20% fetal bovine serum by volume; the pH value is 7.0 to 7.5, and the osmotic pressure is 260 to 300 mOsm / L.

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