阅读说明：本技术 真鲷脑细胞系及其构建方法与应用 (Red porgy brain cell line and its construction method and use ) 是由 罗霞 李宁求 付小哲 林强 牛银杰 刘礼辉 梁红茹 于 2021-09-16 设计创作，主要内容包括：本发明属于生物技术领域,公开了真鲷脑细胞系及其构建方法与应用。该真鲷脑细胞系,名称为真鲷脑BB细胞,于2021年8月12日保藏于位于中国.武汉.武汉大学的中国典型培养物保藏中心,保藏编号为CCTCC NO：C2021227,目前已传至75代,形态以成纤维样细胞为主；该细胞系的建立在丰富鱼类细胞库的同时可为水产动物病毒性疾病的病原分离、培养、检测提供保障,还可为体外基因组RNA干扰、基因敲除提供一个新的研究工具,同时具有作为监测环境污染物的作用。(The invention belongs to the field of biotechnology, and discloses a red sea bream brain cell line, a construction method and application thereof. The red porgy brain cell line is named as red porgy brain BB cell, is preserved in the China center for type culture collection of the university of Wuhan, Wuhan in 2021 at 8 months and 12 days, and has the preservation number of CCTCC NO: c2021227, which has been passed to 75 generations, is predominantly fibroblast-like in morphology; the establishment of the cell line can provide guarantee for pathogen separation, culture and detection of aquatic animal viral diseases while enriching a fish cell bank, also can provide a new research tool for in vitro genome RNA interference and gene knockout, and has the function of monitoring environmental pollutants.)

1. A red sea bream brain cell line is named as red sea bream brain BB cell, which is preserved in the China center for type culture Collection of the university of Wuhan, 2021, 8 months and 12 days, with the preservation number of CCTCC NO: C2021227.

2. the method for constructing a red sea bream brain cell line according to claim 1, comprising the steps of: 1) taking a red sea bream brain tissue, shearing, digesting by adopting collagenase digestive juice containing serum, centrifuging, removing a supernatant, re-suspending by using a proliferation culture solution, and culturing at 25-30 ℃ to obtain primary red sea bream brain cells; 2) after primary red porgy brain cells grow into a monolayer, digesting the monolayer by trypsin, re-suspending the monolayer by a proliferation culture solution, and carrying out subculture to obtain a red porgy brain cell line.

3. The construction method according to claim 2, wherein:

the collagenase digestive juice containing serum is an L-15 culture solution containing 10-20 v/v% of fetal calf serum, 0.1-0.2 w/v% of I-type collagenase, 200-400U/mL of penicillin, 200-400 mu g/mL of streptomycin and 0.5-1.0 mu g/mL of amphotericin B; preferably, the proliferation culture solution is an L-15 culture solution containing 20-30 v/v% of fetal calf serum, 10-20 ng/mL of human epidermal growth factor, 10-20 ng/mL of human basic fibroblast growth factor, 100-200U/mL of penicillin, 100-200 mu g/mL of streptomycin and 0.25-0.5 mu g/mL of amphotericin B and having a pH value of 7.0-7.4.

4. The construction method according to claim 2 or 3, characterized in that:

after the red sea bream brain cells are subcultured to 5 generations in the step 2), the proliferation culture solution is an L-15 culture solution which contains 9-11 v/v% fetal calf serum and has a pH value of 7.0-7.4.

5. The method for culturing the red sea bream brain cell line according to claim 1, comprising digesting the red sea bream brain cell line according to claim 1 with pancreatin, adding a culture medium, and culturing;

the culture medium is at least one of a DMEM culture medium containing fetal calf serum, an L-15 culture medium containing fetal calf serum and an M199 culture medium containing fetal calf serum.

6. The use of the red sea bream brain cell line according to claim 1 in any one of the following (1) to (3);

(1) separating aquatic animal viruses;

(2) culturing aquatic animal viruses;

(3) detection of aquatic animal viruses is a non-diagnostic treatment destination.

7. The use of the red sea bream brain cell line according to claim 1 in screening drugs for preventing or treating diseases caused by viral infection in aquatic animals.

8. Use according to claim 6 or 7, characterized in that:

the aquatic animal virus is at least one of nervous necrosis virus, grouper iridovirus, infectious spleen and kidney necrosis virus, mandarin fish rhabdovirus and tilapia lake virus.

9. The use of the red sea bream brain cell line according to claim 1 for monitoring environmental pollutants.

10. Use according to claim 9, characterized in that:

the environmental pollutant is heavy metal, further cadmium.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a red sea bream brain cell line, and a construction method and application thereof.

Background

The fish cell line is used as an important experimental model, has the obvious advantages of low cost, strong controllability, contribution to analysis of a large number of samples and the like, and is widely applied to the research fields of virology, immunology, toxicology and the like of aquatic organisms. Sensitive cell lines are important carriers for virus research, and pathogenic diagnosis, infection mechanism and immune prevention all need to be based on the cell lines as the premise. Starting from the establishment of the first fish cell line RTG-2 from Wolf, more than 300 fish cell lines were established, on the basis of which more than 40 aquatic viruses were isolated. Meanwhile, the researchers propose that the fish cell line is also a multipurpose tool in the ecotoxicology research, and can replace the fish acute toxicity experiment to evaluate the cytotoxicity of the medicine. Heavy metal pollution of aquatic animals becomes a very common food hygiene problem, cadmium (Cd) is a typical biological accumulative toxic pollutant, widely exists in water environment, destroys an aquatic ecosystem, poses serious threat to growth and development of aquatic organisms and has potential danger to human health. Compared with living fishes, the fish culture cells as the experimental objects polluted by heavy metal have the following advantages: the cell uniformity and the repeatability are good, and the experimental conditions are controllable; the medicine directly acts with cells, and the reaction speed is high; low cost and no need of large-scale culture equipment such as water change, air inflation and the like.

Pagrus major (Pagrus major) is a rare marine economic fish, distributed from the north to the middle of the Atlantic ocean, including China, Indonesia, Japan, Korea, Philippines, and belongs to offshore warm water bottom fish. With the development of industry and the expansion of culture scale, the marine heavy metal pollution and diseases are more and more frequent, viral diseases become a bottleneck of Red sea bream culture, main pathogeny comprises Red sea bream iridovirus (RSIV), Nervous Necrosis Virus (NNV), rockfish iridovirus (SGIV) and other marine fish viruses, the mortality rate is up to 90%, and the economic loss is high. NNV belongs to the family of Nodaviridae (Nodaviridae), has wide host range, and can infect more than 120 kinds of cultured and wild fishes, including marine fishes such as grouper, Pagruidae and Paralichthys olivaceus, as well as freshwater fishes such as Anguilla anguillata (Anguilla anguillata), Parasilurus asotus (Parasilurus asotus) and Mandarin fish. The first cell line to be used for NNV studies was the SSN-1 cell line established by fry of Channa argus (Channastriatus), a 1996 English scholar, and the subsequent sensitive cell lines were derived mainly from fish tissues such as Epinepheluscoides, Latescal carpifer, and Decapter pompano. The establishment of sensitive cell lines not only enhances the sensitivity of NNV diagnosis, but also makes it possible to study the replication and pathogenicity of the virus at the cellular level.

Brain cells, particularly telencephalon and cerebellum, contain a large number of proliferating cells, and therefore, brain tissue of teleost fish is considered by many scholars to be suitable for the establishment of cell lines. In view of this, studies on fish-derived brain tissue cell lines such as the Epinephelus fuscoguttatus cell line BMGB, the Siniperca chuatsi cell line CPB, the Oppenny red sea bream brain cell line OPPB, the red sea bream brain cell line SaB-1, and the hybrid snakehead brain cell line CAMB have been increasingly conducted. As a sensitive host of NNV, the genuine porgy successfully established cell lines mainly come from tissues such as kidney, embryo, tail fin and the like, but the sensitivity to the virus is low or is not mentioned. After NNV infection, diseased fish are mainly characterized by sleepiness and vacuolation of brain and retina; as a target organ of NNV infection, a red sea bream brain tissue cell line is not reported, so that the construction of the red sea bream brain cell line is necessary.

Disclosure of Invention

In a first aspect of the present invention, there is provided a red sea bream brain cell line.

In a second aspect of the present invention, there is provided the method for constructing a red sea bream brain cell line according to the first aspect.

In a third aspect of the present invention, there is provided a method for culturing a red sea bream brain cell line according to the first aspect.

In a fourth aspect of the present invention, there is provided the use of the red sea bream brain cell line of the first aspect.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides a red porgy brain cell line, which is named as red porgy brain BB cell and is preserved in the China center for type culture collection in the university of Wuhan, 8.12.2021, with the preservation number of CCTCC NO: C2021227.

in a second aspect of the present invention, there is provided a method for constructing a red sea bream brain cell line according to the first aspect of the present invention, comprising the steps of: 1) taking a red sea bream brain tissue, shearing, digesting by adopting collagenase digestive juice containing serum, centrifuging, removing a supernatant, re-suspending by using a proliferation culture solution, and culturing at 25-30 ℃ to obtain primary red sea bream brain cells; 2) after primary red porgy brain cells grow into a monolayer, digesting the monolayer by trypsin, re-suspending the monolayer by a proliferation culture solution, and carrying out subculture to obtain a red porgy brain cell line.

Preferably, the collagenase digestive juice containing serum is L-15 culture solution containing 10-20 v/v% of fetal calf serum, 0.1-0.2 w/v% of I-type collagenase, 200-400U/mL penicillin, 200-400 mu g/mL streptomycin and 0.5-1.0 mu g/mL amphotericin B.

Preferably, the proliferation culture solution is an L-15 culture solution containing 20-30 v/v% of fetal calf serum, 10-20 ng/mL of human epidermal growth factor, 10-20 ng/mL of human basic fibroblast growth factor, 100-200U/mL of penicillin, 100-200 mu g/mL of streptomycin and 0.25-0.5 mu g/mL of amphotericin B and having a pH value of 7.0-7.4.

Preferably, the red sea bream brain tissue in step 1) is obtained by the following steps: soaking and disinfecting the red sea bream with 70-75 v/v% alcohol for 2-3 minutes, wiping off mucus on the body surface, and then dissecting under an aseptic condition to take down brain tissue.

Preferably, the method of shearing in step 1) is as follows: and (3) shearing the brain tissue into tissue blocks of 50-100 cubic millimeters in the HBSS equilibrium solution.

Preferably, the step 1) further comprises the following steps after the cutting: centrifuging at 500-600 rpm/min to collect tissue mass, and centrifuging for 2-3 times.

Preferably, the digestion conditions in step 1) are: digesting for 4-8 h at 25-30 ℃.

Preferably, the conditions of the centrifugation in step 1) are: centrifuging at 1000-1400 rpm for 3-7 minutes.

Preferably, after the red sea bream brain cells are subcultured to 5 generations in the step 2), the proliferation culture solution is an L-15 culture solution containing 9-11 v/v% fetal bovine serum and having a pH value of 7.0-7.4.

Preferably, the red sea bream brain cells in step 2) are subcultured to 25 generations (including 25 generations) and are subcultured at a ratio of 1: 2.

Preferably, after the red sea bream brain cells are subcultured to 25 generations in the step 2), the red sea bream brain cells are subcultured according to the ratio of 1: 3.

In a third aspect of the present invention, there is provided a method for culturing the red sea bream brain cell line according to the first aspect of the present invention, comprising digesting the red sea bream brain cell line according to the first aspect of the present invention with pancreatin, adding a culture medium to culture;

the culture medium is at least one of a DMEM culture medium containing fetal calf serum, an L-15 culture medium containing fetal calf serum and an M199 culture medium containing fetal calf serum.

Preferably, the medium is M199 medium containing fetal bovine serum.

Further preferably, the medium is M199 medium containing 10% fetal bovine serum.

In a fourth aspect of the invention, there is provided use of the red porgy brain cell line of the first aspect of the invention.

The application of the red sea bream brain cell line of the first aspect of the invention in culturing aquatic animal viruses.

The application of the red sea bream brain cell line of the first aspect of the invention in the separation of aquatic animal viruses.

The use of a red sea bream brain cell line according to the first aspect of the present invention for the detection of aquatic animal viruses in a non-diagnostic therapeutic setting.

The application of the red sea bream brain cell line in the screening of the medicament is the medicament for preventing or treating diseases caused by virus infection of aquatic animals.

The invention relates to application of the red porgy brain cell line in monitoring environmental pollutants.

Preferably, the aquatic animal virus is at least one of nervous necrosis virus, grouper iridovirus, infectious spleen and kidney necrosis virus, mandarin fish rhabdovirus and tilapia lake virus.

Preferably, the environmental contaminant is a heavy metal, further cadmium.

The invention has the beneficial effects that:

the invention separates the brain tissue cells of the red sea bream by adopting a collagenase digestion method through a large amount of creative labor of an inventor, carries out primary culture, constructs a red sea bream brain cell line for the first time, is named as the red sea bream brain BB cell (BB cell), is preserved in the China center for type culture preservation, CCTCC NO: c2021227, which has been passed to 75 generations, is predominantly fibroblast-like in morphology; virus susceptibility tests show that besides Nervous Necrosis Virus (NNV) and grouper iridovirus (SGIV), BB cells infected by common freshwater fish viruses such as mandarin fish rhabdovirus (SCRV), Infectious Spleen and Kidney Necrosis Virus (ISKNV), tilapia lake virus (TiLV) and the like can generate obvious cytopathic effect; the virus titer obtained after the NNV, the SGIV, the ISKNV, the SCRV and the TiLV infect BB cells of different generations respectively is stable at 8.0-8.3 log TCID₅₀/mL、7.0～7.2log TCID₅₀/mL、4.0～4.2log TCID₅₀/mL、4.3～4.5log TCID₅₀a/mL and a TCID of 4.3 to 4.5log₅₀Per mL; PCR detection further proves that the virus can infect BB cells; toxicology tests show that cadmium can generate obvious cytotoxicity after exposing BB cells for 24 hours, and the BB cell activity is gradually reduced along with the increase of Cd concentration, so that a dose-effect relationship is presented; BB cells of the cadmium exposed group show green and red fluorescence after Annexin and PI double staining, which indicates that cadmium can induce the BB cells to generate obvious apoptosis and necrosis phenomena; the establishment of the cell line can provide guarantee for pathogen separation, culture and detection of aquatic animal viral diseases while enriching a fish cell bank, also can provide a new research tool for in vitro genome RNA interference and gene knockout, and has the function of monitoring environmental pollutants.

Drawings

Fig. 1 is a morphological feature diagram of red sea bream brain cells of different generations: wherein, A is a morphological feature map (4 x) of the first generation of red sea bream brain cells; b is a morphological feature map (4X) of red sea bream brain cells of passage 24.

Fig. 2 is a statistical graph of survival rate of different generations of frozen genuine porgy brain cells after recovery.

FIG. 3 is a diagram showing the result of karyotype analysis of red sea bream brain cells of different generations: wherein, A is the nuclear type analysis statistical result chart of the red porgy brain cells of the 15 th generation; b is a nuclear pattern analysis statistical result chart of 60 th generation red sea bream brain cells; c is metaphase karyotype of red sea bream brain cells at passage 60.

Fig. 4 is a graph of the results of the effect of different media on the growth of red sea bream brain cells: wherein A is a line graph of change of the number of red sea bream brain cells in different culture media along with time; b is a line graph showing the change in the number of red sea bream brain cells with time in M199 medium containing FBS at various concentrations.

Fig. 5 is a graph showing the results of transfection efficiency of red sea bream brain cells (BB cells): wherein A is a result of transfection of BB cells with a eukaryotic expression vector containing pEGFP-N1 by liposome 2000 (20X); b is the BB cell map in bright field (20 ×).

Fig. 6 is a graph showing the effect of the Nervous Necrosis Virus (NNV) on red sea bream brain cells (BB cells): wherein, A is uninfected BB cell map (20 ×); b is the BB cell map 48h after NNV infection (20 ×); c is a map of BB cells 72h after infection with NNV (20X).

FIG. 7 is a graph showing the effect of Epinephelus groupers iridovirus (SGIV) on red sea bream brain cells (BB cells): wherein, A is uninfected BB cell map (10 ×); b is a map of BB cells 24h after infection with SGIV (10 ×); c is a map of BB cells (10X) 48h after infection with SGIV.

FIG. 8 is a graph showing the effect of Infectious Spleen and Kidney Necrosis Virus (ISKNV) on red sea bream brain cells (BB cells): wherein, A is uninfected BB cell map (20 ×); b is BB cell map (20 ×) 48h after infection with ISKNV; c is a map of BB cells (20X) 96h after infection with ISKNV.

Fig. 9 is a graph showing the effect of mandarin fish rhabdovirus (SCRV) on red sea bream brain cells (BB cells): wherein, A is uninfected BB cell map (10 ×); b is a map of BB cells 24h after infection with SCRV (10 ×); c is a map of BB cells (10X) 48h after infection with SCRV.

FIG. 10 is a graph showing the effect of Tilapia Lake Virus (Tilapia Lake Virus, TiLV) on red sea bream brain cells (BB cells): wherein, A is uninfected BB cell map (10 ×); b is the BB cell map 3d after infection with TiLV (10 ×); c is the BB cell map (10X) after infection with TiLV for 4 d.

FIG. 11 is a diagram showing the results of PCR detection of cell fluids infected with different viruses: wherein A is a PCR detection result graph of ISKNV infected cell sap, SCRV infected cell sap and SGIV infected cell sap; b is a PCR detection result graph of TiLV infected cell sap and NNV infected cell sap; in A, M represents Marker 2000, and 1 represents an ISKNV negative control; 2 represents ISKNV infected cell sap; 3 represents the SCRV negative control; 4 represents the cell fluid infected by SCRV; 5 represents SGIV negative control; 6 represents SGIV infected cell fluid; in B, M represents Marker 2000,1 represents a TiLV negative control; 2 represents TiLV infected cell fluid; 3 represents NNV negative control; 4 represents NNV-infected cell sap.

FIG. 12 is a graph showing the effect of Cd concentration on red sea bream brain cell viability.

Fig. 13 is a graph of the effect of Cd exposure on red porgy brain cells: wherein, A is a morphogram (10 x) of differently treated red sea bream brain cells; b is a combined image of bright field, Annexin V-FITC staining, PI staining and Annexin V-FITC/PI staining of red sea bream brain cells 24h after Cd exposure and a control group (20X).

Detailed Description

The present invention will be described in further detail with reference to specific examples.

It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The materials, reagents and the like used in the present examples are commercially available reagents and materials unless otherwise specified.

The reagents used in this example were analytical grade reagents and were commercially available from a regular channel.

Example 1 construction of red sea bream brain cell line

1. Treatment of brain tissue: firstly, healthy red sea bream (purchased from a certain farm in Yangjiang, Guangdong province) with the body length of about 8cm is soaked and disinfected in 75% alcohol for 2 minutes, mucus on the body surface is wiped off by gauze, the red sea bream is placed in an ultraclean workbench to be dissected and taken off brain tissue, the brain tissue is roughly cut into tissue blocks with the size of 50-100 cubic millimeters in a small glass bottle filled with HBSS (hand's Balanced Salt Solution) balance liquid, the tissue blocks are collected at 600 revolutions per minute, and the centrifugation is repeated for 3 times.

2. Primary culture: placing the tissue block into a collagenase digestive fluid containing serum (the digestive fluid formula is L-15 culture fluid containing 10 v/v% fetal calf serum, 0.1 w/v% type I collagenase, 200U/mL penicillin, 200 mu g/mL streptomycin and 0.5 mu g/mL amphotericin B) to digest for 6 hours at 28 ℃, after digestion is finished, centrifuging for 5 minutes at 1200 rpm, collecting the tissue cell suspension subjected to digestion treatment, adding 5 milliliters of special proliferation culture fluid for red sea bream brain cells (the culture fluid formula is L-15 culture fluid containing 20 v/v% fetal calf serum, 10ng/mL human epidermal growth factor, 10ng/mL human basic fibroblast growth factor, 100U/mL penicillin, 100 mu g/mL streptomycin, 0.25 mu g/mL amphotericin B and the pH value of 7.2), the cell suspension was transferred to a 25mL cell culture flask and cultured in an incubator at 28 ℃. Sucking out 2.5mL of culture solution in the 25mL culture bottle every 3-5 days to remove non-adherent tissues and dead cells, and adding 2.5mL of fresh special culture solution for red sea bream brain cells.

3. Subculturing: after the red sea bream brain cells grow into a monolayer (about 21 days), sucking out the culture solution in the culture bottle, cleaning for 2 times by using HBSS (basic hydrogen sulfide system) balance solution, adding 0.5mL of trypsin digestive juice (0.25% trypsin-EDTA solution, GIBCO) for digestion for 1-2 min, sucking out the trypsin digestive juice, adding 10mL of special multiplication culture solution for the red sea bream brain cells, and blowing and beating the bottom of the culture bottle by using a dropper to prepare cell suspension; according to the following steps: 2 (5 mL of brain cell suspension is taken out and added into a new 25mL culture bottle, 10mL of special proliferation culture solution for the red sea bream brain cells is added), and after the cells grow into a monolayer (3-5 days), subculturing is carried out by the same method; after passage to 25 passages, the ratio of 1:3, and subculturing once every 2-3 days. After 5 th generation, the special proliferation culture solution for red porgy brain cells is removed with human epidermal growth factor, human basic fibroblast growth factor, penicillin, streptomycin and amphotericin B, and the fetal calf serum concentration is reduced to 10 v/v%.

The red porgy brain cell line is named as red porgy brain BB cell, is preserved in the China center for type culture collection of the university of Wuhan, Wuhan in 2021 at 8 months and 12 days, and has the preservation number of CCTCC NO: C2021227.

fig. 1 is a morphological feature diagram of red sea bream brain cells of different generations: in FIG. 1, A is the first generation of red sea bream brain cells, which are mainly composed of epithelioid and fibroblast-like cells; in FIG. 1, B is the 24 th generation of red sea bream brain cells, mainly fibroblast-like cells.

Example 2 Properties of Red Pagrus brain cells

1. Cell cryopreservation and recovery

Freezing and storing cells: cells were frozen every 5 passages from passage 5 to passage 65 in culture. When the red sea bream brain cells after subculture grow to logarithmic phase, a programmed cooling box is adopted to be placed in a refrigerator at minus 80 ℃ overnight and then transferred into a liquid nitrogen tank for long-term storage.

Cell recovery: the frozen cells of the 30 th, 40 th, 50 th and 60 th generations were picked, taken out of the liquid nitrogen tank after 6 months, 1 year and 2 years of freezing, and rapidly placed in a 37 ℃ water bath for thawing, the cell suspension was transferred into a cell culture flask, 5mL of M199 medium containing 10% FBS was added, cultured overnight at 28 ℃ and the fresh medium was changed the next day. After the cells were thawed, a small amount of cell suspension was placed in a centrifuge tube, stained with trypan blue, observed under a microscope, and the cell viability was calculated. The results are shown in FIG. 2: after 6 months, 1 year and 2 years of freezing, respectively selecting the freezing cells of the 30 th, 40 th, 50 th and 60 th generations for resuscitation, wherein the cells show the survival rate of 80-90 percent and can grow full in 2-3 days; the cell has been passed for more than 70 times, the cell state is good, the cell can be stably proliferated, and the form has no obvious change, so that it is proved that the red porgy brain cell has become continuous cell line, and is named as red porgy brain cell (red porgy brain BB cell, BB cell) line.

2. Karyotyping analysis

Passing the 15 th and 60 th generation cells to 75cm²The culture flask of (4) was added with colchicine (Sigma) at a final concentration of 0.2. mu.g/mL for about 48 hours, and the culture was continued at 28 ℃ for 4 hours; pancreatin digested cells, resuspended in 0.075M KCl solution, methanol added: glacial acetic acid (3: 1) stationary liquid. Dripping cells by a cold dripping method, and observing and counting under a phase contrast microscope; 100 well-dispersed split phases with good morphology are randomly selected for karyotype analysis. The results are shown in FIG. 3: 98% of the cells at the 15 th generation contain 48 chromosomes, and the number of chromosomes of the 60 th generation cells is distributed in 32-50, wherein the cell chromosome mode is 42 and 48.

3. Cell growth characteristics

(1) Selection of optimal Medium

After digesting the 55 th generation BB cells in the logarithmic growth phase with trypsin, Dulbecco ' modified Eagle's medium (DMEM, Gibico), Leibovitz's medium L-15(Gibico) and M199(Gibico) containing 10% FBS were added to adjust the initial cell concentration to 2X 10⁵One cell/mL of the cells was inoculated into 12-well plates, 1mL of the cells was cultured at 28 ℃, 3 replicates of the cells were counted every 24 hours using a cell counter (Count star), and the average value was obtained. The cells were counted for 7 consecutive days and cell growth curves were plotted. The results are shown in FIG. 4: the cell growth curve is S-shaped, and the cell amount is maximum when the cells are cultured to 3d in L15 and M199 culture media, and is respectively 7.38 multiplied by 10⁵one/mL and 10.54X 10⁵BB cells grow slowly in DMEM medium until the 5d cell amount reaches the maximum value of 4.89X 10⁵one/mL. This revealed that the cells grew rapidly in M199 with a high peak, and that the cells were suitable as an optimal medium for BB cell culture.

(2) Determination of optimal serum concentration

After digesting BB 55 th generation cells in the logarithmic growth phase with trypsin, M199 medium containing 4%, 6%, 8% and 10% FBS was added to adjust the initial cell concentration to 2X 10⁵One cell/mL of the cells was inoculated into 12-well plates, 1mL of the cells was cultured at 28 ℃, 3 replicates of the cells were counted every 24 hours using a cell counter (Count star), and the average value was obtained. Count for 7 consecutive days and draw growth curves. The results are shown in FIG. 4: test with serum addition of 10% and 8% respectivelyGroup, cells reached a peak at 3d and then started to decrease in number; in the test groups with 6% and 4% serum added, the cells reached a peak at 2d and then started to decrease in number. In 4 test groups, 10% of the serum groups had the highest proliferation rate and the highest peak value, so that the addition of 10% of serum to the M199 culture medium was most suitable for the growth of BB cells.

4. Cell transfection

Coli containing pEGFP-N1 was streaked with LA-Kan⁺After the plate and single colony are picked up for amplification culture, endotoxin-free plasmid extraction is carried out according to the instruction of an OMEGA endotoxin-free plasmid extraction kit, 60-generation BB cells are transfected by referring to a liposome transfection method of Invitrogen, and fluorescence is observed by an inverted fluorescence microscope (Leica). The results are shown in FIG. 5: green fluorescence can be observed 24h after the pEGFP-N1 plasmid is transfected into the BB cells, which indicates that the BB cells can be used as a cell receptor for expressing an exogenous gene.

Example 3 susceptibility to infection of Red sea bream brain cells (BB cells) by Nervous Necrosis Virus (NNV), Epinephelus coioides iridovirus (SGIV), Infectious Spleen and Kidney Necrosis Virus (ISKNV), Siniperca Chuatsi Rhabdovirus (SCRV), Tilapia Lake Virus (TiLV)

Taking BB cell of the 55 th generation in logarithmic growth phase, incubating NNV at 0.1MOI and SGIV, SCRV and TiLV at 0.01MOI for 1h at 28 ℃; the ISKNV is inoculated according to 1MOI, incubated for 2h at 28 ℃, then respectively supplemented with M199 containing 2% serum, and continuously cultured at 28 ℃. Observing cytopathic condition every day, and repeatedly freezing and thawing twice at-80 ℃ when CPE reaches 80%, and harvesting virus liquid.

1. Susceptibility to different viruses

(1) Sensitivity of BB cells to NNV

Collecting BB cell of 55 th generation in logarithmic growth phase, inoculating NNV at 0.1MOI, incubating at 28 deg.C for 1h, supplementing M199 containing 2% serum, and culturing at 28 deg.C. Observing cytopathic condition every day, and repeatedly freezing and thawing twice at-80 ℃ when CPE reaches 80%, and harvesting virus liquid. The results are shown in FIG. 6: after BB cells are infected by NNV for 48 hours, the cells shrink and become round; 96h after infection, a large number of cells are disintegrated and shed; while the control cells grew well; BB cells were shown to be sensitive to NNV, and the virus was able to proliferate on BB cells.

Sequentially diluting the harvested NNV virus solution by 10 times with M199 culture solution, and taking 10 times^-4～10^-10The diluted virus solution was inoculated into 96-well plates full of BB cell monolayers (50 th, 55 th, and 60 th generation cells), the final column was used as a control, 8 dilutions were set in parallel, 100. mu.L of each well was incubated at 28 ℃ for 1 hour, and then M199 culture medium containing 2% serum was added. The cells were further cultured in an incubator at 28 ℃ and the number of cells in the lesion was recorded by observation every day. Continuous observation 10d, calculation of TCID by Reed-Muench method₅₀. The virus titer obtained by different generations of cells is stable at 8.0-8.3 logTCID₅₀mL, indicating that the sensitivity of different generations of cells to NNV infection is stable and the titer is high, so that BB cells belong to NNV high-sensitive cell lines.

(2) Sensitivity of BB cells to SGIV

Collecting BB cell of 55 th generation in logarithmic growth phase, inoculating SGIV at 0.01MOI, incubating at 28 deg.C for 1h, adding M199 containing 2% serum, and culturing at 28 deg.C. Observing cytopathic condition every day, and repeatedly freezing and thawing twice at-80 ℃ when CPE reaches 80%, and harvesting virus liquid. The results are shown in FIG. 7: after 24h of BB cells are infected by SGIV, part of the cells are shed; at 48h after infection, cells swell and a large number of cells disintegrate and fall off; while the control cells grew well; BB cells were shown to be sensitive to SGIV, and the virus was able to proliferate on BB cells.

Sequentially diluting the obtained SGIV virus solution by 10 times with M199 culture solution, and taking 10 times^-4～10^-10The diluted virus solution was inoculated into 96-well plates full of BB cell monolayers (50 th, 55 th, and 60 th generation cells), the final column was used as a control, 8 dilutions were set in parallel, 100. mu.L of each well was incubated at 28 ℃ for 1 hour, and then M199 culture medium containing 2% serum was added. The cells were further cultured in an incubator at 28 ℃ and the number of cells in the lesion was recorded by observation every day. Continuous observation 10d, calculation of TCID by Reed-Muench method₅₀. The virus titer obtained by different generations of cells is stable at 7.0-7.2 logTCID₅₀mL, which shows that the sensitivity of different generation cells to SGIV infection is stable and the titer is higherBB cells therefore belong to the SGIV high sensitive cell line.

(3) Sensitivity of BB cells to ISKNV

And (3) taking BB cells of the 55 th generation in the logarithmic growth phase, inoculating the cells with ISKNV according to 1MOI, incubating for 2 hours at 28 ℃, then supplementing M199 containing 2% serum, and continuing to culture at 28 ℃. Observing cytopathic condition every day, and repeatedly freezing and thawing twice at-80 ℃ when CPE reaches 80%, and harvesting virus liquid. The results are shown in FIG. 8: after 48h of infection of BB cells by ISKNV, the BB cells mainly show a certain degree of shrinkage and little rounding; at 96h after infection, infected cells are rounded and swollen, and a small amount of cells are disintegrated and fall off; while the control cells grew well; it was shown that BB cells were sensitive to ISKNV and the virus was able to proliferate on BB cells.

Sequentially diluting the harvested ISKNV virus liquid by M199 culture solution by 10 times, and taking 10 times of the diluted ISKNV virus liquid^-2～10^-8The diluted virus solution was inoculated into 96-well plates full of BB cell monolayers (50 th, 55 th, and 60 th generation cells), the final column was used as a control, 8 dilutions were set in parallel, 100. mu.L of each well was incubated at 28 ℃ for 2 hours, and then M199 culture medium containing 2% serum was added. The cells were further cultured in an incubator at 28 ℃ and the number of cells in the lesion was recorded by observation every day. Continuous observation 10d, calculation of TCID by Reed-Muench method₅₀. The virus titer obtained by different generations of cells is stable at 4.0-4.2 logTCID₅₀and/mL, which shows that the sensitivity of different generations of cells to ISKNV infection is stable and the titer is relatively high, and BB cells belong to an ISKNV moderate sensitive cell line.

(4) Sensitivity of BB cells to SCRV

Collecting BB cell of 55 th generation in logarithmic growth phase, inoculating SCRV at 0.01MOI, incubating at 28 deg.C for 1 hr, adding M199 containing 2% serum, and culturing at 28 deg.C. Observing cytopathic condition every day, and repeatedly freezing and thawing twice at-80 ℃ when CPE reaches 80%, and harvesting virus liquid. The results are shown in FIG. 9: 42-48 hours after the SCRV infects BB cells, most cells become round, and phenomena of wiredrawing, falling off and plaque occur among the cells; while the control cells grew well; BB cells were shown to be sensitive to SCRV, and the virus was able to proliferate on BB cells.

The harvested SCRV virus solution is sequentially made 10 times by M199 culture solutionSerial dilution, taking 10^-2～10^-8The diluted virus solution was inoculated into 96-well plates full of BB cell monolayers (50 th, 55 th, and 60 th generation cells), the final column was used as a control, 8 dilutions were set in parallel, 100. mu.L of each well was incubated at 28 ℃ for 1 hour, and then M199 culture medium containing 2% serum was added. The cells were further cultured in an incubator at 28 ℃ and the number of cells in the lesion was recorded by observation every day. Continuous observation 10d, calculation of TCID by Reed-Muench method₅₀. The virus titer obtained by different generations of cells is stable at 4.3-4.5 logTCID₅₀and/mL, which indicates that the sensitivity of different generations of cells to the infection of the SCRV is stable and the titer is relatively high, and BB cells belong to a SCRV moderate sensitive cell line.

(5) Sensitivity of BB cells to TiLV

Collecting BB cell of 55 th generation in logarithmic growth phase, inoculating TiLV with 0.01MOI, incubating at 28 deg.C for 1h, adding M199 containing 2% serum, and culturing at 28 deg.C. Observing cytopathic condition every day, and repeatedly freezing and thawing twice at-80 ℃ when CPE reaches 80%, and harvesting virus liquid. The results are shown in FIG. 10: after 3 days after the TiLV infects BB cells, the cells begin to shrink and become round, and a small amount of cells fall off; 4d, disintegrating and falling off a large number of cells after infection, wherein the cells are in a fish-breaking net shape; while the control cells grew well; BB cells were shown to be sensitive to TiLV, and the virus was able to proliferate on BB cells.

Sequentially diluting the harvested TiLV virus liquid by 10 times of M199 culture solution in series, and taking 10 times of the diluted liquid^-2～10^-8The diluted virus solution was inoculated into 96-well plates full of BB cell monolayers (50 th, 55 th, and 60 th generation cells), the final column was used as a control, 8 dilutions were set in parallel, 100. mu.L of each well was incubated at 28 ℃ for 1 hour, and then M199 culture medium containing 2% serum was added. The cells were further cultured in an incubator at 28 ℃ and the number of cells in the lesion was recorded by observation every day. Continuous observation 10d, calculation of TCID by Reed-Muench method₅₀. The virus titer obtained by different generations of cells is stable at 4.3-4.5 logTCID₅₀and/mL, which indicates that the sensitivity of different generations of cells to TiLV infection is stable and the titer is relatively high, and BB cells belong to a TiLV moderate sensitive cell line.

PCR detection of BB cells infected with NNV, SGIV, ISKNV, SCRV, TiLV

Viral genomes are extracted from the harvested virus liquid respectively, primers are designed according to an NNV capsid protein sequence registered by GenBank, Main Capsid Protein (MCP) sequences of SGIV and ISKNV, a mandarin rhabdovirus G protein sequence and a tilapia lake virus Segment 3 sequence, and PCR amplification is carried out (see Table 1). The results are shown in FIG. 11: can be amplified to single bands of 562bp, 317bp, 372bp, 491bp and 430bp, and the sizes of the bands are consistent with the sizes of expected fragments; sequencing the PCR product, wherein the result is consistent with the sequences of NNV capsid protein registered by GenBank, MCP of SGIV and ISKNV, mandarin rhabdovirus G protein and tilapia lake virus Segment 3, and the homology is 100 percent, which shows that the virus can successfully infect BB cells.

TABLE 1 PCR amplification primer sequences

Example 4 toxicology study of Red Pagrus brain cells (BB cells)

Determination of BB cell Activity

The density is 2 x 10⁵BB 55 th generation at one/mL was seeded in 96-well cell culture plates, cultured overnight at 28 ℃ and washed 2 times with PBS. Adding into each well (180 μ L of medium M199 containing 5% serum +20 μ LCdCl)₂) According to the pre-experimental result, the final concentration of Cd respectively reaches 6 concentration gradients of 0.5, 0.1, 0.05, 0.01, 0.005 and 0.001mol/L, 200 mu L of M199 culture medium containing 5% serum is directly added into a control group, 4 multiple wells are arranged on each Cd concentration group and the control group, BB cells after Cd exposure in a 96-well culture plate are collected after culture in an incubator at 28 ℃ for 24 hours, and the cell viability is measured by adopting an MTT method. The data obtained by the experiment are all expressed as mean plus or minus standard deviation (mean plus or minus SD), the data are processed by SPSS 22.0 statistical software, the difference is significant when the P is less than 0.05 and the difference is significant when the cell viability is subjected to the inter-group difference analysis by utilizing t test<The time difference was significant at 0.01. The results are shown in FIG. 12: BB cell viability decreased gradually with increasing Cd concentration and presented a dose-effect relationship. When the concentration of Cd is higher than 0.001mol/L, the treatment group and the control groupThe difference is extremely remarkable (P)<0.01)。

BB apoptosis assay

The density is 2 x 10⁵BB 55 th generation at one/mL was seeded in 12-well cell culture plates, cultured overnight at 28 ℃ and washed 2 times with PBS. According to the preliminary experimental results, according to (900. mu.L of M199 medium containing 5% serum + 100. mu.LCdCl)₂) In the method, a final concentration of 0.1mg/L Cd was selected for 24h exposure of BB cells, and 1000. mu.L of M199 medium containing 5% serum was directly added to the control group. And (3) carrying out apoptosis detection on the BB cells after Cd exposure according to the specification of an Annexin-V-FLUOS staining kit. The results were observed under an inverted fluorescence microscope immediately after staining. The results are shown in FIG. 13: after the cadmium is exposed to BB cells for 24 hours, the cells are partially contracted and rounded by an inverted microscope, most of the cells are rounded and shed after 48 hours, and the cells of a control group are unchanged; after Annexin and PI double staining, BB cells exposed for 24h by cadmium show green and red fluorescence; control group BB cells did not fluoresce red or green. The above results show that cadmium can cause BB cells to generate obvious apoptosis and necrosis phenomena.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Zhujiang aquatic research institute of Chinese aquatic science research institute

<120> red sea bream brain cell line, construction method and application thereof

<130>

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<170> PatentIn version 3.5

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