CN113957037B

CN113957037B - Perch gill cell line, construction and application method thereof

Info

Publication number: CN113957037B
Application number: CN202111382035.2A
Authority: CN
Inventors: 孙云; 周永灿; 魏曹莹; 曹贞洁; 吴莹; 李建龙; 张冬冬
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2023-04-28
Anticipated expiration: 2041-11-22
Also published as: CN113957037A

Abstract

The invention relates to a humpback bass gill cell line, construction and an application method thereof, belonging to the technical field of cell biology, wherein the cell line is preserved in China general microbiological culture collection center (CGMCC No. 23097) of university of Wuhan, china at the date of 22 and 10 in 2021. The invention also provides a preparation method of the humpback bass gill cell line, which can be stably cultured after 15 generations, and the humpback bass gill cell line is stably transferred for 35 generations at present; the cell line prepared by the method can be placed in liquid nitrogen for long-term storage, and normal culture can be continued after recovery. The humpback bass gill cell line can be used for nerve necrosis virus detection and related pathology research, and exogenous plasmid transfection related experiments.

Description

Perch gill cell line, construction and application method thereof

Technical Field

The invention belongs to the technical field of cell biology, and particularly relates to a humpback bass gill cell line, construction and an application method thereof.

Background

The construction of the rainbow trout gonad cell line reported by Wolf et al in 1960 is the earliest of fish cell culture, and up to now, nearly 300 different tissue cell lines of the freshwater fish are established. With the continuous development of biotechnology, fish cell lines have been successfully used in a variety of research fields such as fish virology, toxicology, physiology, immunology, genetic breeding, etc.

The common name of the humpback bass (Cromileptes altivelis), mouse spots, belongs to the subfamilies of the bass, grouper and the genus humpback bass, and is mainly distributed in tropical areas of the Indian ocean and the Pacific ocean. The fish meat is tender and smooth, delicious and delicious, has high nutritive value, is the most rare grouper at present, and has selling price of 800-1000 yuan/kg in hong Kong, guangzhou and other places in China. In recent years, with the breakthrough of artificial breeding technology, the humpback bass gradually realizes large-scale production. However, with the continuous expansion of the production scale, bacterial diseases such as vibrio vulnificus and vibrio harveyi and viral diseases such as iridescence and nerve necrosis continuously burst, and huge losses are caused for the industrial aquaculture of the humpback bass. The research on the immune mechanism and pathogenic bacteria pathogenic mechanism of the humpback bass is significant for analyzing the pathogenic mechanism of the humpback bass and developing related vaccine development work. However, compared with other groupers, the price of the humpback bass is extremely high, the requirements on the culture environment are extremely severe, and the research cost of carrying out in-vivo experiments is extremely high. Therefore, constructing a humpback bass cell line, and developing related researches by using the cell line becomes one of effective methods for solving the problems, and can also provide experimental materials for virus separation, identification and functional gene research.

Disclosure of Invention

The invention aims to provide a humpback bass gill cell line, a construction method and an application method thereof.

The invention is realized by adopting the following technical scheme:

a humpback bass gill cell line which is preserved in China general microbiological culture collection center (CGMCC No. 23097) of university of Wuhan, china at the date of 22 and 10 in 2021.

The invention also provides a construction method of the humpback bass gill cell line, which comprises gill tissue sampling, primary culture, subculture, cell cryopreservation and resuscitating;

the gill tissue is sampled: under the aseptic state, the gill tissue of the humpback bass is sheared, and is cleaned by an L15 culture medium containing penicillin and streptomycin, and then is sheared into small blocks;

the primary culture: sticking the gill tissue block of the humpback bass on the culture surface of a cell culture bottle, lightly reversing the cell culture bottle by 180 degrees, adding a normal-temperature complete culture medium, reversing after 3-5 hours, and enabling the culture medium to be in contact with the tissue block; culturing at 25-28deg.C; after 8-10d gill cells migrate from the primordia and replace 1/3-1/2 of the primordia with fresh complete medium after 3-4 d.

The subculture comprises the following steps:

1) Pouring out the original culture medium when the primary cultured gill cells are fully paved at 85% -95% of the cell bottle bottom, carefully cleaning the primary culture medium for 2-3 times by using sterile PBS, adding 0.25% pancreatin containing EDTA at room temperature, standing and digesting for 1-2min, blowing for 1min by using a liquid transfer device, microscopically observing the cell rounding, adding the complete culture medium, repeatedly blowing, and suspending the cell;

2) Collecting culture solution containing cells, centrifuging at 1000-1200g for 5-8min, performing passage at 1:2, adding complete culture medium, and performing subculture in a constant temperature incubator at 26 ℃;

3) After the cells are passaged to 15 generations, the cells are passaged at a ratio of 1:3, and the cells are passaged for 4-5 days once;

the cells are frozen and thawed:

1) The preparation components of the frozen culture solution and the volume ratio are 70% of L15 culture medium, 10% of DMSO and 20% of fetal bovine serum; the L15 culture medium contains 400IU/ml penicillin and 400 mug/ml streptomycin;

2) After the cells are cultured to the logarithmic phase, pouring out the original culture medium, washing with sterile PBS, adding EDTA-containing 0.25% pancreatin at room temperature, standing for digestion for 1-2min, blowing with a pipette for 1min, adding 4-5ml complete culture medium, repeatedly blowing, suspending cells, centrifuging for 5-8min at 1000-1200g, removing pancreatin, adding the prepared frozen culture solution, lightly blowing with a straw to make the cells uniform, counting, and regulating the final density of the cells in the frozen culture solution to 5×10 ⁶ /ml～5×10 ⁶ Per ml, freezing gill-containing cellsTransferring the storage liquid into a sterilized freezing storage tube;

3) Placing the freezing tube in a freezing box containing isopropanol, then placing in a refrigerator at-80 ℃ for 12-24 hours, and then transferring the freezing tube into liquid nitrogen for long-term storage;

4) Cell resuscitation is to take out a freezing tube in liquid nitrogen, rapidly placing the tube in sterile water at 37 ℃ for water bath melting, transferring the melted cells into a cell culture bottle, adding a complete culture medium, placing the cell culture bottle in a constant temperature incubator at 26 ℃ for static culture, discarding the supernatant after the cells are attached, and adding a fresh complete culture medium.

The invention also provides application of the humpback bass gill cell line.

Compared with the prior art, the invention has the beneficial effects that:

the preparation method of the humpback bass gill cell line is disclosed for the first time, and is simple and feasible; after 15 times of passage, the cell line can be stably cultured, and 35 generations of stable culture are carried out at present; the cell line prepared by the method can be placed in liquid nitrogen for long-term storage, and normal culture can be continued after recovery. The humpback bass gill cell line can be used for detecting nervous necrosis virus and researching related pathology and exogenous plasmid transfection related experiment

Drawings

FIG. 1 is a resuscitating and passaging of a humpback bass gill cell line; resuscitates a cells, passage b

FIG. 2 shows the virus infection of the gill cell line of the jeldrake bass for 5 days and for 7 days in the negative control of FIG. a;

FIG. 3 is a diagram of transfection of the humpback bass gill cell line.

Detailed Description

The technical scheme of the present invention is further explained by examples below, but the scope of the present invention is not limited in any way by the examples.

Example 1 preparation method of a humpback Perch gill cell line

1. Tissue sampling

1) Soaking healthy humpback bass in 15-30ppm potassium permanganate for 20min, killing after anesthesia, wiping the fish body with 75% medical alcohol, wrapping the fish body with gauze soaked with 75% alcohol, and carrying the fish body into a sterile operation room;

2) Shearing part of gill tissue from gill part of fish body with sterilized scissors, washing with L15 culture medium containing 400IU/ml penicillin and 400 μg/ml streptomycin for 3 times, and cutting tissue block into 1mm×1mm small blocks;

2. primary culture

1) The 9 gill tissue blocks are carefully placed on the culture surface of a cell culture bottle, the cell culture bottle is gently inverted for 180 degrees, 3ml of normal-temperature complete culture medium is added, and after 4 hours, the culture medium is carefully inverted to be in contact with the tissue blocks.

2) The 50ml system complete medium formulation was 10ml of fetal bovine serum, 2ml of green streptomycin (penicillin concentration 400IU/ml and streptomycin concentration 400. Mu.g/ml), 500. Mu.l of 100 XNaCl solution filtered through a 0.22 μm filter (0.266 g/ml NaCl in 10L15 medium), 250. Mu.l of Hepes (1M), 37.25ml of L15 medium, and after mixing, placed at 4℃for use.

3) Slightly inverting the cell culture flask with the tissue block to 180 degrees, adding 5ml of normal-temperature complete culture medium, carefully inverting after 5 hours to enable the culture medium to be in contact with the tissue block, and then placing the cell culture flask into a constant-temperature incubator at 26 ℃ for culture;

4) After 10d of culture, after gill cells migrate from the original tissue, replacing 1/2 of the original culture medium with the complete culture medium for 4d, and then placing the original culture medium into an original incubator for culture;

3. subculture

1) When gill cells are fully cultured and flat-bottomed by 90%, taking out the rest tissue blocks, pouring out the original culture medium, carefully cleaning the tissue blocks for 2-3 times by using sterile PBS, adding 1ml of 0.25% pancreatin (containing EDTA) preheated to room temperature in advance, standing and digesting for 2min, blowing for 1min by using a pipette, microscopic observation of cell rounding, adding 5ml of complete culture medium, repeatedly blowing for suspending cells, centrifuging for 5min at 1100g, carrying out passage at 1:2, adding complete culture medium to 4ml per bottle, and carrying out passage culture in a constant-temperature incubator at 26 ℃.

2) After the cells are passaged to 15 generations, passaging can be carried out at a ratio of 1:3, and passaging is carried out for about 4 days once;

4. cryopreservation of cells: selecting gill cell line with vigorous growth and exponential growth phase, 25cm ² Cell culture flask 2 flask, 1ml of 0.25% pancreatin pre-warmed to room temperature in advance was added(containing EDTA), standing and digesting for 2min, blowing with a pipette for 1min, microscopic observation of cell rounding, adding 5ml of complete culture medium, repeatedly blowing, suspending cells, centrifuging for 5min at 1100g, suspending cells with 1ml of cell freezing solution (L15 culture medium containing 10v/v% dimethyl sulfoxide, 20v/v% fetal calf serum and 80% green streptomycin), transferring to a cell freezing tube, placing the freezing tube in a cell freezing box, adding isopropanol, and transferring the freezing tube to a liquid nitrogen tank for preservation after 24 h.

5. Cell resuscitation: taking out the stored freezing tube from the liquid nitrogen tank, rapidly placing in sterile water at 37 ℃ for water bath melting, transferring the melted cells into a cell culture bottle, adding 4ml of complete culture medium, placing in a constant temperature incubator at 26 ℃ for static culture, discarding the supernatant after the cells are attached, and adding 4ml of fresh complete culture medium. As shown in fig. 1.

Example 2 experiment of viral infection in the Perch gill cell line

1. The cultured humpback bass gill cell line is transferred into a 6-hole plate, and 500 μl of complete culture medium is added to culture cells for adherent growth for 3d.

2. Old medium was discarded, washed twice with PBS, and complete medium containing the nerve necrosis virus and complete medium with equal volume of PBS added to each group of three parallel wells were added. Culturing in a constant temperature incubator at 26 ℃ for 7d.

3. The cells after incubation are placed under an inverted microscope for daily observation and photographing

4. As shown in figure 2a, the cells of the negative infection group (uninfected virus) grew well; as shown in FIGS. 2 b and c, positive infected groups (infectious viruses) formed significant lesions in cells around 5d and 7d.

The detection result shows that the humpback bass gill cell line can be used for detecting nervous necrosis virus and researching related pathology.

Example 3 Green fluorescent protein eukaryotic expression plasmid vector pEGFP-N3 transfection experiment of the humpback Perch gill cell line

1. Inoculating the humpback bass gill cells to a 6-hole plate, adding 2ml of complete culture medium to culture the cells, and transfecting when the cells are paved at 70-90% of the hole bottom for 48 hours;

2. the 6-well plate was washed 2-3 times with sterilized PBS before transfection, 2ml of serum-free medium formulation (50 ml system) of penicillin (penicillin concentration 400IU/ml and streptomycin concentration 400. Mu.g/ml), 500. Mu.l of 100 XNaCl solution filtered through a 0.22 μm filter (0.266 g/ml NaCl in 10L15 medium), 250. Mu.l of Hepes (1M), 47.25ml of L15 medium were added per well.

3. Preparing a transfection solution:

1) In the transfection kit

3000 reagent 3.75 μl, 125 μl +.>

Diluting the culture medium, and uniformly mixing to prepare solution A;

2) 5 μl transfection kit P3000 ^TM Reagent, 10. Mu.l of pEGFP-N3 plasmid (plasmid addition 800-1.6.5. Mu.g) and

125 mu l of culture medium and vortex shaking for 2-3s to prepare solution B,

3) Mixing the solution A and the solution B uniformly to prepare a transfection mixed solution, carrying out vortex oscillation for 10s, and standing at room temperature for 15min.

4. The mixed solution is gently dripped into gill cells to be transformed containing 2ml of fetal calf serum culture solution,

after 6h, changing to a complete culture medium;

after 5.3d, the medium was poured off, the 6-well plates were washed 3 times with PBS, fixed with 4% paraformaldehyde for 2h, and photographed under an inverted fluorescence microscope.

6. As shown in FIG. 3, the cells can see green fluorescence under an inverted fluorescence microscope, and the transfection efficiency can reach more than 50%.

The detection result shows that the humpback bass gill cell line can be used for exogenous plasmid transfection related experiments.

Claims

1. A humpback bass gill cell line, which is characterized in that the cell line is preserved in China general microbiological culture collection center (CGMCC No. 23097) of university of Wuhan, china at the time of 10 months and 22 days of 2021.

2. The use of the humpback bass gill cell line according to claim 1, wherein the use is in the detection of a neural necrosis virus infection and in the transfection of an exogenous plasmid, said use being for non-disease diagnosis purposes.