CN113025574A

CN113025574A - Micropterus salmoides brain cell line and application thereof

Info

Publication number: CN113025574A
Application number: CN202110363152.8A
Authority: CN
Inventors: 袁军法; 戴彩姣; 余利; 李莉娟
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2021-06-25
Anticipated expiration: 2041-04-02
Also published as: CN113025574B

Abstract

The invention discloses a micropterus salmoides brain cell line and application thereof, wherein the micropterus salmoides brain cell line is preserved in China Center for Type Culture Collection (CCTCC) at 03-25.2021, and the preservation numbers are as follows: CCTCC NO: C202166. The brain cell line MSBr of the micropterus salmoides has stable passage, is successfully passed for more than 65 generations at present, has good growth state, has wide application range, can be used for virus separation identification, pathogen function research, environmental pollutant detection, gene screening, function analysis, fish tumor research and the like, can be used for monitoring, diagnosis and epidemiological investigation and research of aquatic animal viral diseases, development of diagnostic kits of related viral diseases, vaccine development and the like, and has great application value and social and economic values.

Description

Micropterus salmoides brain cell line and application thereof

Technical Field

The invention belongs to the technical field of disease prevention and control of aquatic organism cells and aquaculture, and particularly relates to a micropterus salmoides brain cell line and application thereof.

Background

Cell culture studies of aquatic animals (mainly fish) were later than mammals, starting in the 60's of the 20 th century. In 1962, Woft and Quimby established the first fish cell line in the world, the rainbow trout gonad cell line. Since then, the research on fish cell culture is very rapid, and 275 strains of fish cell lines have been reported all over the world. The culture of fish cells in China starts at the end of the 70 th 20 th century, and a diploid cell line of the snout tissue of the grass carp established by Zhangnianji and the like is the first fish cell line in China. The number of fish cell lines established at present is about 70, and most of the fish cell lines are from marine fishes. The cell strain is used as an in vitro material with bioactivity and has the advantages that the culture and maintenance of the cells do not need large-scale culture equipment and the cost is low; the size of the cell individual is uniform, the genetic background is very similar, the experimental conditions can be accurately controlled, and the reproducibility is good. Therefore, the cell line of fish is an important material and model for virology research, environmental poison detection, physiology, immunology, gene function analysis, cell engineering breeding, germplasm preservation and other researches. Although the establishment technology of primary fish cells is mature at present, great contingency and difficulty exist for obtaining a virus-sensitive continuous cell line, so that the establishment of a target fish cell line needs preparation of a large batch of primary cell strains so as to screen out a sensitive cell line, which is time-consuming and labor-consuming.

Micropterus salmoides (Micropterus salmoides), also known as Lateolabrax japonicus, belong to Perciformes, Sunglyudae and Lateolabrax, are originally sourced from the Missippi river system of California, USA, and are introduced into the world due to their fresh and delicious meat quality, strong disease resistance, rapid growth, easy capture and wide temperature range. Chinese introduces Japanese sea Perch in Guangdong Buddha in 1983, and successfully breeds in 1985. After more than 40 years of development, the micropterus salmoides become important economic breeding varieties in China. However, with the increasing degree of artificial intensification, the breeding density is continuously increased, and the disease layer of the micropterus salmoides is endless, especially the disease of virus. Rhabdoviruses and iridoviruses are the major etiological agents of weever viral diseases in california. In recent years, rhabdovirus diseases of micropterus salmoides are greatly outbreaked in the breeding process of micropterus salmoides seedlings, 80 percent of ponds are diseased seriously, the death rate can reach 90 percent in a short time, the medicine application effect is poor, and the economic loss is huge.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a micropterus salmoides brain cell line and application thereof, the cell line has stable passage, is successfully passed for more than 65 generations at present, has good growth state, has wide application range, can be used for virus isolation identification, pathogen function research, environmental pollutant detection, gene screening, function analysis, fish tumor research and the like, can be used for monitoring, diagnosis and epidemiological investigation research of aquatic animal viral diseases, development of diagnostic kits of related viral diseases, vaccine development and the like, and has great social and economic values.

One of the objectives of the present invention is to provide a brain cell line of micropterus salmoides, which has been preserved in the China center for type culture Collection at 03/25/2021, with the addresses of: the preservation number of the brain cell line of the micropterus salmoides is as follows: CCTCC NO: C202166.

Further, the culture temperature of the brain cell line of the micropterus salmoides is 28 ℃.

Furthermore, in the culture process of the brain cell line of the micropterus salmoides, the volume concentration of fetal calf serum is 10% or 15%.

The second object of the present invention is to provide a method for constructing the brain cell line of micropterus salmoides, which comprises:

s1, narcotizing micropterus salmoides, draining blood, soaking in 75% alcohol for 2min, and taking brain tissue of micropterus salmoides under aseptic condition;

s2, rinsing the brain tissue with sterile PBS buffer solution, and soaking in AIM culture solution for 30 min;

s3, cutting the brain tissue into 1mm³Size, adding trypsin, and digesting at 28 deg.C for 20 min;

s4, stopping digestion by using M199 culture medium containing 20% FBS by volume concentration, passing the obtained cell suspension through a cell mesh screen, and centrifuging the filtered cell suspension at 1000r/min for 7 min;

s5, removing supernatant after centrifugation, and utilizing inclusion bodiesResuspending and precipitating with 20% FBS M199 culture medium to obtain resuspension mixture, adding into cell culture flask, and culturing at 28 deg.C and 5% CO₂Culturing in an incubator to obtain primary micropterus salmoides brain cells, digesting with trypsin when the monolayer cells reach a fusion state, and performing subculture to obtain micropterus salmoides brain cell lines.

Further, in step S4, the cell suspension is passed through a 70 μm cell sieve, and simultaneously, the remaining tissue fragments on the surface of the cell sieve are pressed by a syringe.

The invention also aims to provide the application of the brain cell line of the micropterus salmoides in the proliferation culture of aquatic animal viruses and/or the development of virus vaccines.

Further, the aquatic animal virus comprises: micropterus Salmoides Rhabdovirus (MSRV), Spring Viremia of Carp Virus (SVCV), Grass Carp Reovirus (GCRV), frog iridovirus (RGV), Red-spotted grouper neuronecrosis virus (RGNNV), Infectious Hematopoietic Necrosis Virus (IHNV).

The fourth purpose of the invention is to provide the application of the brain cell line of the micropterus salmoides in the expression of exogenous genes.

The fifth purpose of the invention is to provide the application of the brain cell line of the micropterus salmoides in screening of anti-aquatic animal virus drugs.

The sixth purpose of the invention is to provide the application of the brain cell line of the micropterus salmoides in biological models of drug evaluation, gene screening and function analysis, pathogen function research, cell engineering breeding and environmental poison detection.

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

(1) the brain cell line of the micropterus salmoides has stable passage, can be passaged for at least 65 generations, is sensitive to various common aquatic animal viruses such as MSRV, SVCV, GCRV and the like, and can be applied to the aspects of pathogen infection mechanism and pathogen function research, development of related diagnostic preparations, separation and identification of unknown fish viruses and the like.

(2) The micropterus salmoides brain cell line has high titer of viruses such as MSRV, GCRV and the like, can be used for large-scale culture of related viruses, or preparation of attenuated vaccines, inactivated vaccines and the like, or screening and evaluation of anti-aquatic virus drugs, and further realizes prevention and control of related viral diseases.

(3) The cell line of the invention has single cell type and controllable culture conditions, so the cell line has uniform reaction to pollutants, good repeatability of experimental results, small error and convenient observation, detection and recording; and the cells are directly contacted with the pollutants, so that the using amount of the pollutants is relatively reduced, the environmental pollution is avoided, the cell reaction can be visually observed, the damage part and the toxic action target spot of the environmental pollutants can be researched on the cell or sub-cell level by combining the technologies of an electron microscope and the like, and the toxicity of the environmental pollutants can be evaluated, namely, the cell line can be used for replacing the live fish in the research fields of fish toxicology, environmental pollutant or toxic detection and the like.

(4) The micropterus salmoides brain cell line can efficiently express exogenous genes, so the cell line can be used as a cell model for gene screening and function analysis, such as screening antiviral genes of micropterus salmoides and research and analysis of gene functions.

Drawings

FIG. 1 is a graph showing the results of observing cell morphology at different growth generations in example 1 of the present invention, wherein FIG. 1-A shows a 5 th generation micropterus salmoides brain cell, FIG. 1-B shows a 10 th generation micropterus salmoides brain cell, FIG. 1-C shows a 20 th generation micropterus salmoides brain cell, and FIG. 1-D shows a 60 th generation micropterus salmoides brain cell;

FIG. 2 is a graph showing the growth curves of the MSBr cell line at different culture temperatures in example 2 of the present invention;

FIG. 3 is a graph showing the growth curves of the MSBr cell line at different fetal bovine serum concentrations in example 2;

FIG. 4 is a standard growth curve of the cell line MSBr in example 2 of the present invention;

FIG. 5 shows the chromosome analysis results of the cell line MSBr in example 2 of the present invention, in which FIG. 5-A shows the chromosome number statistics and FIG. 5-B shows the karyotype analysis results;

FIG. 6 shows the results of the sensitivity test of the cell line MSBr to 7 viruses in example 3 of the present invention.

FIG. 7 shows the results of PCR qualitative detection of the corresponding viruses in cells after 7 viruses are inoculated on the cell line MSBr in example 3 of the present invention.

FIG. 8 shows the TCID of MSBr-sensitive virus as a cell line in example 3 of the present invention₅₀Measuring results;

FIG. 9 is a graph showing the results of detection of fluorescent protein expression after transfection of pEGFP-N1 with cell line MSBr in example 4 of the present invention;

FIG. 10 shows the results of the test of the application of the MSBr cell line in the antiviral drug screening in example 5 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 isolation and culture of brain cells of Perch

1. Experimental reagents and materials

(1) Experimental animals: the molecular method is used for repeatedly verifying that the healthy micropterus salmoides without being infected with aquatic animal viruses are about 10cm in size, and the micropterus salmoides offspring field from Zhijiang city in Hubei province are cultured in a greenhouse culture barrel of an aquiculture institute base of Huazhong agricultural university.

(2) Experimental apparatus: scalpel, ophthalmic scissors, ophthalmic forceps, 70 μm cell mesh screen, syringe, alcohol cotton, gauze, and the like.

(3) Experimental reagent: AIM culture medium, liquid medium M199, trypsin-EDTA, penicillin/streptomycin 10000U/mL, Fetal Bovine Serum (FBS) were purchased from Gibco; amphotericin B, gentamicin, colchicine were purchased from Biosharp; dimethyl sulfoxide (DMSO) was purchased from Sigma, and Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF) were purchased from Peprotech.

(4) Preparing a culture solution:

primary culture solution: mixing 80mL of M199 culture solution, 20mL of FBS, 1mL of penicillin/streptomycin (10000U/mL), 100. mu.L of EGF (10. mu.g/mL) and 100. mu.L of FGF (10. mu.g/mL), and storing at 4 ℃;

subculture solution: 85mL or 90mL of M199 culture solution, 15mL or 10mL of FBS mixture, 1mL of penicillin/streptomycin (10000U/mL), and storing at 4 ℃;

PBS buffer: 8g NaCl, 0.2g KCl, 1.44g NaHPO₄，0.24g KH₂PO₄Dissolving in triple distilled water, adjusting pH to 7.2, adding volumetric flask to 1L, and sterilizing under high pressure.

2. Primary culture

The detected healthy micropterus salmoides are temporarily cultured in a laboratory for 10 days, and the experiment is started after no disease is detected, and the specific steps are as follows:

(1) before the experiment, the fish is anesthetized by MS-222, and blood is drained as much as possible; soaking in 75% ethanol for 2min, and taking out brain tissue in ultra-clean bench under aseptic condition;

(2) rinsing brain tissue with sterile PBS buffer solution for 2 times, and soaking in appropriate amount of AIM culture solution for 30 min;

(3) cutting brain tissue into 1mm³Adding 3-5 mL of trypsin-EDTA, and digesting at 28 ℃ for 20 min;

(4) after the digestion was terminated with complete medium (20% FBS + M199 medium by volume), the resulting cell suspension was passed through a 70 μ M cell screen and the remaining tissue fragments on the surface of the cell screen were gently pressed with a syringe, followed by centrifugation of the filtered cell suspension at 1000r/min for 7 min;

(5) centrifuging, removing supernatant, resuspending the precipitate with complete culture medium (20% FBS + M199 culture medium), adding the mixture into T25 cell culture flask, and placing at 28 deg.C and 5% CO₂Culturing in an incubator, observing the adherence and growth condition of the cells every day, digesting with trypsin when the monolayer cells reach a fusion state, and carrying out subculture.

3. Subculturing

Sucking out old culture solution in the culture bottle when 70-80% of primary cells are fully paved at the bottom of the bottle; adding 1mL of 0.25% trypsin-EDTA solution into the bottle, standing and digesting for 5 min; observing under a microscope, after most cells are shortened and become round, sucking out trypsin, continuously digesting by using the residual trypsin and tapping a culture bottle, and adding a fresh culture medium to stop digestion after part of the cells are seen to be detached; gently blowing off white fog-shaped adherent cells by glass bent tube, gently mixing to obtain single cell suspension, adding appropriate amount of culture medium, recording cell name, generation number and date, standing at 28 deg.C and 5% CO₂Culturing in the incubator; after the cells grow to fill the bottom of the bottle, the cells are subjected to trypsin digestion again and are subjected to scale-up culture according to the growth rate of the cells.

The primary culture solution is used for the early cell passage, the passage culture solution containing 15% FBS is more stable for the cells after 6 generations, and the passage culture solution containing 10% FBS is used after 10 generations.

4. Freezing and thawing

Freezing and storing cells: when the cells in the T25 cell culture flask were in logarithmic growth phase and the cell monolayer reached about 80% of the bottom of the flask, the cells were cryopreserved. Removing the original culture medium by suction, adding 1mL of 0.25% trypsin-EDTA solution for digestion, adding 1mL of cell cryopreservation solution (900 mu L of fetal bovine serum +100 mu L of LDMSO) to prepare cell suspension, adding the cell suspension into a cryopreservation tube, placing the cell suspension into a cryopreservation box after marking correspondingly, after overnight at-80 ℃, transferring the cell suspension into a liquid nitrogen tank for preservation at (-196 ℃) and making related records.

Cell recovery: the frozen cells are taken out from the liquid nitrogen tank, and are rapidly transferred into a constant-temperature water bath at 37 ℃ to be continuously shaken to be rapidly melted, and the mouth of the freezing tube is not submerged into water in the shaking process so as to avoid cell pollution. After the ice in the freezing tube is melted, sucking out the cell suspension by a pipette, transferring the cell suspension into a 1.5mL centrifuge tube, centrifuging for 10min at 1000r/min, removing the supernatant to remove DMSO in the freezing solution, gently blowing and resuspending the cells by using 1mL complete culture medium of 20% fetal calf serum, transferring the cells into a new T25 cell culture bottle, supplementing a proper amount of culture medium, placing the bottle at 28 ℃ and 5% CO₂Culturing in constant temperature incubator, observing every dayMorphology and growth of the cells after recovery.

And selecting a cell line which can continue to stably proliferate and grow after passage for 10 times, freezing and storing the cell line and recovering the cell line. It was named Micropterus salmoides brain cell line (MSBr) and has been deposited at the chinese type culture collection at 03 months and 25 days 2021 at the address: the preservation number of the Wuhan university in Wuhan City of China is as follows: CCTCC NO: C202166. Cell recovery was performed on the obtained cell lines at the 10 th and 30 th generations according to the above method, and it was found that the cells still had strong proliferation ability and good state,

the brain tissue cell line MSBr is passaged 1 time every 3 days, and the morphology of the cells under different growth generations is observed, and the result is shown in figure 1, wherein figure 1-A is 5 th generation micropterus salmoides brain cells, figure 1-B shows 10 th generation micropterus salmoides brain cells, figure 1-C shows 20 th generation micropterus salmoides brain cells, and figure 1-D shows 60 th generation micropterus salmoides brain cells. According to the culture conditions of the above subculture, it was observed that each cell grew vigorously, and through continuous culture for more than 400 days, it had been successfully subcultured for more than 65 generations, and the growth state was good at present.

Example 2 optimal culture Environment, growth Curve and chromosome analysis of brain cells of Perch

1. Optimum culture temperature of brain cell line MSBr of micropterus salmoides

Selecting four different culture temperatures of 16, 22, 28, and 37 deg.C, adjusting cell density to 1 × 10 using M199 culture medium containing 10% FBS⁵One cell per mL, the cell suspension was inoculated at 500. mu.L/well into 12-well plates and placed in 4 incubators at different incubation temperatures. 3-well cells were taken out of each experimental group every 1d, collected by Trypsin-EDTA digestion and counted, and cultured for 5d in total, and counted 5 times in succession, and the growth curves of the cells at different culture temperatures were plotted as shown in FIG. 2. The results show that the brain cell line MSBr of the micropterus salmoides can grow at four temperatures, wherein 28 ℃ is the optimal culture temperature of the MSBr cells.

2. Optimal fetal bovine serum concentration of brain cell line MSBr of micropterus salmoides

Respectively preparing FBS with 2%, 5%, 10% and 15% volume concentrationM199 culture solution, adjusted cell density to 1X 10⁵Each of 4 fetal bovine serum-concentrated media was inoculated at 500. mu.L/well into 12-well plates and cultured in an incubator at 28 ℃. 3-well cells were taken out from each experimental group every 1d, collected by Trypsin-EDTA digestion and counted, co-cultured for 5d, counted 5 times in succession, and a growth curve was prepared. The cell growth curves at different fetal calf serum concentrations are shown in FIG. 3. The results show that under the 28 ℃ culture condition, the proportion of the fetal calf serum is increased from 2 percent to 15 percent, and the growth rate of the MSBr cells is increased along with the increase of the proportion of the fetal calf serum, wherein the volume concentration of the fetal calf serum is optimal when the fetal calf serum is 10 percent or 15 percent.

3. Standard growth curve of brain cell line MSBr of micropterus salmoides

Taking 25 th generation Lateolabrax japonicus brain cells, digesting with Trypsin-EDTA, and adjusting cell density to 1 × 10⁵Each of the cells was inoculated into a 12-well plate at 500. mu.L/well, and cultured in an incubator at 28 ℃. Taking out 3 holes of cells every day, collecting the cells by a Trypsin-EDTA digestion method, counting, co-culturing for 7 days, continuously counting for 7 times, drawing a growth curve and calculating the doubling time of a cell population, wherein the result is shown in figure 4, and shows that the first 2d of the adherent growth of MSBr cells is a retardation phase, the 2 nd to 5 th are logarithmic growth phases, the 5 th to 6 th are stationary phases, and the 6 th later stage enters a decline phase. The population doubling time of the brain cells of the micropterus salmoides is 49.6h by calculation, which indicates that the growth and division potential of the brain cells of the subcultured micropterus salmoides are still very vigorous.

4. Chromosome analysis of brain cell line MSBr of micropterus salmoides

Adding colchicine with final concentration of 15 mug/mL into MSBr cells of 26 th generation in logarithmic growth phase, treating at 28 ℃ for 4h, and collecting cells; performing hypotonic treatment on 6-7mL of ice water for 30min, and centrifuging at 1000r/min for 7 min; pre-fixation for 2min with 2mL of fixative (V methanol: V glacial acetic acid ═ 3:1), followed by addition to 5mL, fixation for 15 min; centrifuging at 1000r/min for 7min to remove supernatant, adding 5mL of stationary liquid, and fixing for 15 min; the tablets were dropped by the cold drop method, dried and stained with 5% Giemsa for 30 min. The results are shown in FIG. 5. By counting the chromosomes of 80 brain cells with clear and well-dispersed metaphase micropterus salmoides, wherein the figure 5-A is the statistical result of the chromosome number, the result shows that the MSBr chromosome number distribution is from 34 to 62, and the cell with the chromosome mode of 48 accounts for 47.5 percent of the total counted cells. The karyotype analysis results are shown in fig. 5-B, and the karyotype formula is 2 n-48-22 m +8sm +18t, i.e. there are 11 sets of centromere chromosomes (m), 4 pairs of sub-centromere chromosomes (sm), and 9 pairs of end centromere chromosomes (t) in the MSBr cell chromosomes.

Example 3 sensitivity testing of the brain cell line MSBr of Micropterus salmoides to different viruses

1. Sensitivity experiment of brain cell line MSBr of micropterus salmoides to different aquatic animal viruses

The cell density is adjusted to 1 × 10 after the cell is digested by Trypsin-EDTA⁵one/mL, in an amount of 1 mL/well, was seeded in 6-well plates. When the cells just grow on the pore plate, after being washed twice by PBS buffer solution, 7 viruses of different viruses (largemouth black bass rhabdovirus MSRV, carp spring viremia virus SVCV, grass carp reovirus GCRV, frog iridovirus RGV, red spotted grouper nervous necrosis virus RGNNV, infectious hematopoietic necrosis virus IHNV and infectious spleen and kidney necrosis virus ISKNV) with 1MOI are inoculated to the brain cell line MSBr of the largemouth black bass of the invention, and the inoculation amount is 200 muL. After inoculation, cells were observed to produce CPE, and cells not inoculated with virus were used as a control (NC), and the results are shown in FIG. 6; and qualitative detection was performed by PCR, the results are shown in FIG. 7. The results show that obvious cytopathic effect (CPE) appears in the cells inoculated with the viruses MSRV, SVCV, GCRV, RGV, RGNNV and IHNV, corresponding viral genes are detected in the cells, and the cells inoculated with the virus ISKNV have no obvious cytopathic effect and have no corresponding genes detected in the cells, which indicates that the brain cell line of the Lateolabrax japonicas cannot proliferate the virus ISKNV and can proliferate the viruses MSRV, SVCV, GCRV, RGIHV, RGNNV and NV, and provides a powerful material for related research of aquatic animal viruses.

2. TCID of sensitive virus inoculated MSBr cells₅₀Measurement of

In order to compare the proliferation effect of MSBr on 6 viruses such as MSRV, SVCV, GCRV, RGV and the like, MSBr cells which grow well are digested, sampled and counted according to the principle of measuring the virus titer by a Reed-Muench method,the cells were cultured in M199 liquid medium at 1X 10⁵The cells were diluted at a concentration of one/mL and seeded into 96-well plates at an initial amount of 100. mu.L/well for 24h to allow sufficient adherence. On the next day, the cell plate was removed and the growth medium was discarded, 100. mu.L of M199 maintenance solution containing 5% FBS was added to each well, then the virus samples to be tested were added to 2 to 11 rows of wells of the 96-well plate in the order of dilution from low to high at 100. mu.L/well, 100. mu.L/well was added to 1 and 12 rows, and the M199 maintenance solution containing 5% FBS was used as a negative control, and the experiment was repeated 2 times. Cell plates were incubated at 28 ℃ with 5% CO₂Observing cytopathic effect (CPE) after culturing in an incubator, recording the end point, and determining the TCID of different viruses according to the principle of determining the virus titer by a Reed-Muench method₅₀The results are shown in table 1 and fig. 8.

TABLE 1 TCID of MSBr-sensitive virus cell line₅₀Measurement results

The results showed that the proliferation efficiency of the 6 viruses was 10^4.495To 10^7.665TCID₅₀ml^-1Wherein the propagation efficiency of GCRV virus is 10 at most^7.665TCID₅₀ml^-1Second, virus SVCV with a proliferation efficiency of 10^6.665TCID₅₀ml^-1。

Example 4 transfection experiment of brain cell line MSBr of Perch in Micropterus salmoides

Digesting 28 th generation MSBr cells by using Trypsin-EDTA to adjust the cell density to 1 multiplied by 10⁵And (4) inoculating the cells per mL in a 6-well plate according to the amount of 2mL per well, and starting transfection after 80-90% of each well of the cells is filled. A clean sterile centrifuge tube was added with 200. mu.L of Opti-MEM medium, and 2.0. mu.g of pEGFP-N1 plasmid were added, as 1: 2 and 1: 3 Add 4. mu.L FishTrans or 6. mu.L PEI transfection reagent, gently pipette and mix well, and stand at room temperature for 20 min. The 6-well plate with cells cultured therein was replaced with 2mL of fresh culture medium (containing fetal bovine serum) per well, and 200. mu.L of the transfection reagent-pEGFP-N1 mixture per well of the six-well plate was added dropwise to the entire wellAfter this time, PEI transfection was performed by the same procedure with gentle mixing. After further culturing for 24 hours, the GFP fluorescent fusion protein was observed, and the results of detection of the fluorescent protein expression are shown in FIG. 9. The results show that after 24h of transfection, MSBr cells can observe strong green fluorescence signals, which indicates that GFP gene can be promoted to be expressed by CMV promoter, and MSBr cell line can express exogenous genes. The transfection efficiency is determined by a fluorescence microscope observation method, and the detection result shows that the transfection efficiency can be up to more than 25%.

Example 5 application of brain cell line MSBr of Micropterus salmoides in antiviral drug screening

Taking micropterus salmoides rhabdovirus MSRV as an example, the brain cell line MSBr of micropterus salmoides is used for verifying the effect of mangiferin and taurine in inhibiting the MSRV virus.

1. Application of mangiferin in treatment or prevention of MSRV

Inoculating a largemouth bass brain cell line MSBr into a 6-well plate, after a monolayer grows to 70-80%, sucking away the culture solution, respectively adding complete culture solution containing 0, 12.5, 25, 50 and 100 mu g/mL mangiferin and PBS for pretreatment for 8h, sucking away the culture solution, adsorbing cells by using 100 mu L MSRV virus with the concentration of 0.1MOI, incubating for 1h at 28 ℃, washing off free virus, continuously adding complete culture medium containing corresponding concentration mangiferin and PBS, and carrying out incubation for 1h at 28 ℃ and 5% CO₂The incubator was continued for 48h (three independent replicates were set for each concentration treatment). And collecting cells after 48 hours to extract total RNA, and performing fluorescent quantitative detection on the MSRV-G gene expression level after reverse transcription. The primer pair for quantitatively detecting the MSRV-G gene comprises the following components:

F：CCGTCCAAACTAGCAACAT；

R：ATTCGGGTTATCGGTGGC。

the quantitative determination results are shown in FIG. 10. The result shows that when the concentration of mangiferin is 12.5 mug/ml, the MSRV-G gene is greatly reduced (p is less than 0.01), and is reduced by 69.24% compared with the control group; and along with the increase of the concentration, the antiviral effect is more obvious, wherein the mangiferin group with the concentration of 25 mu g/ml is reduced by 80.49 percent compared with the control group, the mangiferin group with the concentration of 50 mu g/ml is reduced by 89.74 percent compared with the control group, and the mangiferin group with the concentration of 100 mu g/ml is reduced by 90.06 percent compared with the control group.

2. Application of taurine in treatment or prevention of MSRV

Inoculating a micropterus salmoides brain cell line MSBr into a 6-well plate, after the brain cell line MSBr grows to a single layer of 70-80%, sucking and discarding the culture solution, pretreating with a complete culture solution of 0, 12.5, 25, 50, 100 mu g/mL taurine and PBS for 6h, sucking and discarding the culture solution, adsorbing cells by 100 mu L of MSRV virus with the concentration of 0.1MOI, incubating for 1h at 28 ℃, washing off free virus, continuously adding a complete culture medium containing taurine and PBS with corresponding concentrations, and carrying out 28 ℃ and 5% CO₂The incubator was continued for 24h (three independent replicates were set for each concentration treatment). And collecting cells after 24h to extract total RNA, and performing fluorescent quantitative detection on the MSRV-G gene expression level after reverse transcription. The quantitative determination results are shown in FIG. 10. The results show that when the concentration of taurine is 12.5 mug/ml, the expression of the MSRV-G gene is obviously reduced (p is less than 0.05), and the expression is reduced by 37.32 percent compared with the control group. When the concentration of taurine is 25 mug/ml and 50 mug/ml, the expression level of the gene is greatly reduced (p is less than 0.01), and is respectively reduced by 61.34 percent and 72.16 percent compared with the control group. However, when the concentration of taurine was 100. mu.g/ml, the gene expression level was reduced by only 46.05% as compared with the control group, i.e., taurine inhibited the replication of MSRV but did not have concentration dependence.

The experiments prove that the micropterus salmoides brain cell line MSBr can be used for screening aquatic animal virus-resistant drugs, namely the cell line MSBr can be used as an inhibitor or drug for screening animal models and inhibiting virus proliferation, and provides a powerful material for diagnosis of aquatic animal virus diseases and screening and evaluation of aquatic animal virus-resistant drugs so as to efficiently prevent and control related virus diseases.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Claims

1. A micropterus salmoides brain cell line, wherein the micropterus salmoides brain cell line has a deposit number of: CCTCC NO: C202166.

2. The brain cell line of micropterus salmoides according to claim 1, wherein the brain cell line is cultured at a temperature of 28 ℃.

3. The brain cell line of micropterus salmoides according to claim 1, wherein the volume concentration of fetal bovine serum in the culture process of micropterus salmoides is 10% or 15%.

4. The method of constructing a brain cell line of micropterus salmoides according to claim 1, wherein the method comprises:

s5, removing supernatant after centrifugation, carrying out heavy suspension precipitation by using an M199 culture medium containing 20% FBS (20% FBS by volume) to obtain a uniformly suspended solution, adding the uniformly suspended solution into a cell culture flask, and carrying out 5% CO treatment at 28 DEG C₂Culturing in an incubator to obtain primary micropterus salmoides brain cells, digesting with trypsin when the monolayer cells reach a fusion state, and performing subculture to obtain micropterus salmoides brain cell lines.

5. The method of claim 4, wherein the cell suspension obtained in step S4 is passed through a 70 μm cell sieve, and simultaneously, a syringe is used to press the remaining tissue fragments on the surface of the cell sieve.

6. The use of the brain cell line of micropterus salmoides according to claim 1 in the propagation culture of aquatic animal virus and/or development of virus vaccine.

7. The use of claim 6, wherein the aquatic animal virus comprises: largemouth black bass rhabdovirus, carp spring viremia virus, grass carp reovirus, frog iridovirus, red spotted grouper nervous necrosis virus and infectious hematopoietic necrosis virus.

8. Use of a micropterus salmoides brain cell line according to claim 1 for the expression of exogenous genes.

9. The use of the brain cell line of micropterus salmoides according to claim 1 in screening of anti-aquatic animal virus drugs.

10. The use of the brain cell line of micropterus salmoides of claim 1 as a biological model for drug evaluation, gene screening and functional analysis, pathogen function research, cell engineering breeding, and environmental toxicant detection.