CN118028216A - A fin ray cell line of Brandt's pomfret and its construction method and application - Google Patents

Abstract

The invention discloses a fin ray cell line of Brandt's pomfret, the deposit number of which is CCTCC NO: C2023386, the cell line has a fast cell proliferation rate, can be continuously passaged and remain stable. The invention discloses a method for constructing the cell line, and also discloses the application of the fin ray cell line of Brandt's pomfret in expressing exogenous genes and immunodetection of viral stimulants.

Description

Trachinotus brothers fin line and construction method and application thereof

Technical Field

The invention belongs to the technical field of fish cell culture, and relates to a trachinotus brothers fin cell line, a construction method and application thereof.

Background

Buchinotus ovatus (Trachinotus blochii), commonly known as golden pomfret, belongs to the order of Perciformes, the genus Trachinotus, the family of Trachinotus, and the domestic cultivation yield of 2021 is 24.39 ten thousand tons, and becomes one of the first three varieties of mariculture fishes. Jin Chang is favored in the aquaculture industry as a marine fish variety with important commercial value and development prospect due to delicious taste and rich nutrition. In recent years, as the cultivation scale of golden pomfret is enlarged and the cultivation density is increased, the occurrence of fish diseases is caused, the sustainable development of the cultivation industry is restricted, and the damage of viral diseases, especially the epidemic of nervous necrosis viruses and the like, to golden pomfret is most common and serious for a long time. Therefore, the research on disease control and fine variety breeding of the trachinotus brothers is urgent.

A great number of researches show that the fish cells are good vectors for researching fish viruses, and are mainly used for scientific researches such as virus separation and identification, virus sensitivity, virus pathogenesis and the like. The research of fish cell culture starts in the early 60 s of the 20 th century, wolf and Quimby established the rainbow trout (Oncorhynchus mykiss) gonad cell line RTG-2, after which the research of fish cell line developed rapidly. The fish cell culture is widely applied to researches in aspects of gene function analysis, virus research, toxicology, endocrinology, germplasm resource protection, genetics and the like, and is an important research means.

The technology for establishing the fish cell line is relatively mature at present, but great difficulty still exists in obtaining a seawater fish cell line suitable for stable passage of an experimental model.

Disclosure of Invention

The invention aims to provide a trachinotus brucei fin line, a construction method and application thereof, and establishes the trachinotus brucei fin line by using a tissue migration method, wherein the cell proliferation speed of the cell line is high, the cell line can be continuously passaged and kept stable, and the invention also provides the application of the trachinotus brucei fin line in immunodetection of expressing exogenous genes and virus stimulators.

The technical scheme adopted by the invention for realizing the purposes is as follows:

The invention provides a trachinotus brothers fin cell line with a preservation number of CCTCC NO: C2023386.

The invention also provides a construction method of the trachinotus brothers fin cell line, which comprises the following steps:

(1) Treatment of fin structure: cleaning fin of young trachinotus brucei with L-15 basal medium, shearing in L-15 basal medium, cleaning with cell screen, inoculating to bottom of cell culture flask, and slowly adding L-15 complete medium to the other side of cell culture flask for culturing.

(2) Primary culture: fresh L-15 complete medium was changed every 2-3 days during primary culture, and cells were removed from the tissue mass on day 7 of primary culture.

(3) Subculture: after the confluence of the fin cells of the pompano reaches 90%, sucking out the old culture medium, adding PBS for cleaning once, digesting the adherent cells for about 2min by using 0.25% pancreatin containing EDTA, observing the cells under an inverted microscope to round and start falling off, adding a complete cell culture medium to stop digestion, lightly blowing and dispersing the suspended cells, and carrying out cell splitting and subculturing.

Preferably, the L-15 basal medium contains 400U/mL of a mixture of green streptomycin, the L-15 complete medium contains 20% Fetal Bovine Serum (FBS), 400U/mL of a mixture of green streptomycin, and 0.01% recombinant human basic fibroblast growth factor (bFGF).

Preferably, in the step (1), the culture flask is placed in a 28 ℃ incubator for inversion culture, and the culture flask is slowly turned over and placed in the incubator after being stuck for about 6 hours.

Preferably, in the step (3), cell separation is carried out according to the ratio of 1:2, and the cells are placed in a 28 ℃ incubator for subculture.

The invention also provides application of the trachinotus brothers fin cell line in immunodetection of expression exogenous genes and virus stimulators.

Further, trachinotus brothers fin cells were efficiently transfected with exogenous expression plasmids via FISHTRANS.

Further, when the trachinotus brothers fin cells are stimulated by poly (I: C) for 12h, the expression level of IFN system related genes mita, irf3, irf, if, vig1 and isg15 in the fin cells is obviously increased.

The invention has the beneficial effects that:

The invention establishes a trachinotus brothers fin line by using a tissue migration method, has high cell proliferation speed, can continuously passage and keep stable, and also provides application of the trachinotus brothers fin line in expressing exogenous genes and virus stimulus immunodetection, thereby providing important experimental materials for developing pathogenesis of nervous necrosis viruses and the like and research on diagnosis and prevention and control technologies in future, and enriching cell resources of trachinotus brothers.

Drawings

FIG. 1 is a photograph of establishment and subculture of a trachinotus brothers fin cell line; part A of the graph shows the cell morphology of the 0 th generation of the trachinotus brothers fin cells which migrate on day 7 under a microscope; part B is the monolayer cell morphology of the cell of the first generation fin bar of subculture on the 10 th day; parts C and D are cell morphology at passage 20 and passage 50 of subculture after fin cell resuscitation.

FIG. 2 shows PCR amplification of the 18S rRNA and 16S rRNA genes of trachinotus brothers fin cells; parts A and B in the figure are amplified fragments and sequences of the 18S rRNA gene of the fin cell; parts C and D are amplified fragments and sequences of the 16S rRNA gene of the fin cell.

FIG. 3 shows the growth characteristics of trachinotus brothers fin cells; part A of the diagram is a fin cell growth optimal culture medium type screening diagram; part B is a fin cell growth optimum serum concentration screening chart.

FIG. 4 is a graph showing metaphase chromosome number and split phase morphology of trachinotus brothers fin cells; parts a and B of the graph are the number and morphology of the 8 th generation fin cell chromosomes; parts C and D are the number and morphology of the 51 th generation fin cell chromosomes.

Fig. 5 is a cell cycle analysis of trachinotus brothers fin cells.

FIG. 6 shows the expression of green fluorescent protein in the fin cells of the 50 th generation trachinotus brothers; part a of the graph shows the effect of FISHTRANS transfection reagents on green fluorescent protein transfection in fin cells; part B is the effect of the FuGENE HD transfection reagent on green fluorescent protein transfection in fin cells.

FIG. 7 shows IFN-related gene expression after stimulation of a trachinotus brothers fin cell line with poly (I: C).

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and with reference to the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.

Examples

1. Construction of a Trachinotus brothers fin cell line

(1) Treatment of fin structure: placing a young trachinotus brothers (about 80g in weight) with the age of about 6 months into a 10L barrel, adding iodine, soaking for sterilization for 15 minutes, dripping 2-3 drops of eugenol for anesthesia, then wiping the surface of the trachinotus brothers with 75% alcohol cotton balls, quickly cutting fin strips by a sterile transfer into an ultra-clean workbench, cleaning 2-3 times with a L-15 basal medium [ L-15 (Solarbio) with 400U/mL of a green streptomycin mixed solution ], cutting into pieces in the L-15 basal medium, cutting the fin strip tissues into tissue small blocks with the size of 1mm ³ by using ophthalmic scissors, cleaning by using a 100-mesh cell screen, then inoculating to the bottom of a 25cm ² culture flask, slowly adding about 5mL of L-15 complete medium [ L-15 (Solarbio) with 20% Fetal Bovine Serum (FBS), 400U/mL of a green streptomycin mixed solution, slowly turning over a dry culture flask when the culture flask is reversely placed at 28 ℃, culturing in a left-right side of a dry culture flask, and slowly turning over the culture flask when the culture flask is reversely placed for 6 times.

(2) Primary culture: cells migrate from the tissue mass on day 7 of primary culture by tissue migration (see FIG. 1A); fresh complete medium was changed every 2-3 days during primary culture, and the specific procedures were: discarding the old culture medium, and replacing the fresh complete culture medium; subculturing can be performed when the cell confluency reaches 90%.

(3) Subculture: after the fin cells of the pompano are fully paved with a monolayer (the confluence of cells is about 90%), sucking the old culture medium by a sterile pipette, adding PBS for cleaning once, digesting the adherent cells by 0.25% pancreatin containing EDTA for about 2min, when the cells are observed to be round and start to fall off under an inverted microscope, adding the complete culture medium of the cells to stop digestion, lightly blowing the dispersed suspension cells, separating the cells according to 1:2, leading the complete culture medium to be 5 mL/bottle, putting the culture medium into a 28 ℃ incubator for subculture, and carrying out subculture for about 3-4 days. Fig. 1B shows the cell morphology of the 1 st generation trachinotus ovatus fin.

(4) Cell cryopreservation and resuscitation: collecting cell suspension after pancreatin digestion, centrifuging, discarding supernatant, adding 500 μl of precooled cryopreservation liquid (comprising 10% DMSO and 90% fetal calf serum) to resuspend cells, transferring the cell suspension into a cell cryopreservation tube, sequentially cooling at-20deg.C for 1h and-80deg.C overnight, transferring into liquid nitrogen, and preserving for a long time to complete cell cryopreservation; taking out from liquid nitrogen, rapidly placing on ice to melt, transferring into a constant-temperature water bath kettle at 28 ℃ to shake until the ice is melted, adding a cell complete culture medium, centrifuging to remove the culture medium and frozen stock solution, adding the cell complete culture medium to resuspend cells, and uniformly spreading in a culture bottle to culture to finish cell resuscitate; the survival rate of the resuscitated cells is more than 80%. FIG. 1C shows that cryopreserved 20 th generation fin cells after 48h recovery can be plated to about 90% of the flasks.

2. Determining that the constructed fin cell line is derived from trachinotus brothers

(1) And taking 6 th generation cells with stable growth and good morphology, digesting with 0.25% trypsin, inoculating the 6 th generation cells into a large dish, and after 24 hours, spreading the cells on the bottom of the culture dish by 90%, and carrying out digestion and centrifugation to collect the cells.

(2) DNA from the collected fin cells was extracted using tissue DNA kit (TIANGEN). Specific primers are designed according to the genome of the pomfret brucei to amplify the 18S rRNA and 16S rRNA genes, wherein the primers of the 18S rRNA and the 16S rRNA are respectively as follows: amplification primers for the 18S rRNA gene (18S rRNA-F:5'-TACAGGACTCTTTCGAGGCCCTGT-3' and 18S rRNA-R:5'-CTTGCGCC GGTCCAAGAATTTCAC-3'; fragment size 455 bp) and amplification primers for the 16S rRNA gene (16S rRNA-F:5'-CAAAGGTAGCGTAATCACTTGTCT-3' and 16S rRNA-R:5'-TATGGACTCTAAAAGGGGATTGCG-3'; fragment size 418 bp) (FIGS. 2A, C). In addition, the obtained PCR products were detected by agarose gel electrophoresis and sent to a company for sequencing, and sequence alignment analysis showed that the 18S rRNA and 16S rRNA sequences were 100% similar to the 18S rRNA and 16S rRNA sequences of T.blocii on known NCBI, respectively, indicating that the fin-bar cells were derived from T.blocii (FIGS. 2B, D).

The trachinotus brothers fin cell line GPF Trachinotus blochii has been preserved in China Center for Type Culture Collection (CCTCC) at the date of 28, 12, 2023, with the preservation number of CCTCC NO: c2023386, the preservation address is: chinese university of Wuhan and Wuhan.

3. Determining the optimal culture medium type and the optimal serum concentration for constructing the trachinotus brothers fin cell line

(1) Taking 30 th generation cells with stable growth and good morphology, digesting with 0.25% trypsin, inoculating the cells into a six-hole plate, inoculating the cells into each hole of a culture plate at an initial density of 1X 10 ² cells/mL, sucking out a primary culture medium after the cells are attached to the wall and are stable, cleaning by PBS, respectively culturing the cells into L-15 (Solarbio), M199 (ZOMANBIO) and DMEM (biosharp) complete culture mediums, adding 20% FBS, 400U/mL of a green streptomycin mixed solution and 0.01% recombinant human alkaline fibroblast growth factor into each complete culture medium, and culturing at 28 ℃. Cells were collected from each of the 3 wells on days 1,2, 3, and 4 of three media, the cells were digested and the cell count was counted under a microscope using a blood cell counting plate, the experiment was repeated 3 times, and a growth state diagram of the cell line under different media types was plotted (fig. 3A).

(2) The 30 th generation cells with stable growth and good morphology are taken, digested by 0.25% trypsin and inoculated into a six-hole plate, inoculated into each hole of a culture plate at an initial density of 1X 10 ² cells/mL, respectively prepared into complete L-15 culture mediums with FBS concentration of 10%, 15% and 20%, the cells are cultured by using the three serum concentration L-15 complete culture mediums at 28 ℃, the cells of each hole of the three culture mediums of days 1,2,3 and 4 are taken, 3 holes are collected from each experimental group, the cells are digested and the cell number is counted by using a blood cell counting plate under a microscope, the experiment is repeated 3 times, and the growth state of the cell line under different FBS concentrations is plotted (figure 3B). The results showed that the growth rate of cells increased as the concentration of FBS in the L-15 medium increased from 10% to 20% at the same medium type L-15 and culture temperature. Considering the practical effect of FBS and the high cost, the fetal bovine serum concentration can be suitably reduced to 15% at the time of subculture, particularly after passage 50.

4. Chromosome nuclear analysis of trachinotus brothers fin cell line

Chromosome karyotype analysis is carried out on fin cells of the 8 th and 51 th-generation trachinotus ovatus, and the specific steps are as follows:

(1) After the fin-bar cells are fully paved at more than 90% of the bottom of the culture dish, replacing a fresh culture medium, adding colchicine with the final mass concentration of 1 mug/mL into the culture medium, and incubating the culture medium in a constant temperature incubator at 28 ℃ for 12 hours;

(2) Discarding the original culture medium, washing twice with PBS, treating the cells with trypsin digestion solution, collecting the cells after the cells are completely removed from the wall, and transferring the cells into a 10mL centrifuge tube;

(3) Centrifuging at 350g for 10min, discarding supernatant, suspending in 8mL 0.075M KCl, blowing with gun head, mixing cell precipitate, and hypotonic at 37deg.C for 50min;

(4) 2mL of pre-cooled Carnot's fixative (in the ratio of methanol: acetic acid=3:1, as-is) at 4 ℃ is slowly added into a 10mL centrifuge tube, and pre-fixed for 5min at room temperature;

(5) Centrifuging the cell suspension again, discarding the supernatant, blowing and mixing the cell precipitate with 3mL of Kano's fixing solution precooled at 4 ℃, fixing for 15min at room temperature, and repeating the operation for 2-3 times;

(6) According to the cell quantity, suspending the cell in 200-500 mu L of Carnot's fixative;

(7) The tablet is dripped by adopting a cold dripping method. Dropping the cell suspension on a pre-cooled glass slide at the temperature of minus 20 ℃ at a height of more than 120 cm;

(9) After the baking sheet machine is baked, dyeing for 30min by using a Jim Sa dyeing kit, washing with clear water, naturally airing and sealing sheets;

(10) Chromosomes in mid-term and well dispersed were searched under a 100-fold oil microscope and observed and photographed.

Chromosome number and karyotype are the basis of cytogenetics, and are important indicators for identifying biological species, sex, and the like. As a result of the above chromosome analysis, the fin cells of the 8 th-generation trachinotus ovatus were subjected to chromosome analysis, and the fin cells were spread in the middle stage as normal diploids, and the count showed that the number of chromosomes was varied between 33 and 52, and that more than 32% of the cells had model values of 48 chromosomes at the 8 th generation (FIGS. 4A-B). Similarly, more than 1/3 of the 51 st-generation trachinotus brothers fin cells had 48 chromosomes (FIGS. 4C-D). In conclusion, the cell line obtained by the invention has the same chromosome number as that of the trachinotus brothers, accords with the chromosome number characteristics of the trachinotus brothers, and has stable chromosome number in the passage process.

5. Cell cycle analysis of the trachinotus brothers fin cell line of the invention

Cell cycle of the 48 th generation fin cells was analyzed. 8mL of cells (2X 10 ⁴ cells/mL) were inoculated into a 10cm cell culture dish, cultured at 28℃for 24 hours, and centrifuged at 350g for 10min after digestion of the fin cells with pancreatin, washing the fin cells with PBS, and centrifuging to remove the supernatant. 1mL of DNA staining solution (Multisciences, beijing, CHN) was added to the fin cell pellet, and the fin cells were resuspended by shaking for 10 seconds. The fin cells were then incubated at room temperature for 30min and fluorescent signals were detected with CytoFLEX (Beckman). The data was analyzed using FlowJo software with the following results: the population of proliferating cells can be classified into G0-G1 phase (DNA pre-synthesis phase, blue), S phase (DNA synthesis phase, green), G2 phase and M phase (mitosis phase, red). The histogram shows two distinct peaks at the G0/G1 and G2/M phases (FIG. 5A). The proportion of mitotic cells was 4.26%, the proportion of G0-G1 phase cells was 61.1% and the proportion of S phase cells was 26.0% irrespective of cell debris in the cultured cells (FIG. 5B).

6. The trachinotus brothers fin cell can express exogenous gene by transfection

Fin cells were transfected with pEGFP-N3 plasmid expressing Green Fluorescent Protein (GFP), and their transfection efficiency and gene expression were examined to determine whether they were suitable for studying viral pathogenesis and formulating antiviral strategies. Fin cells passaged 50 times were added to a coverslip in a 6-well plate at a concentration of 2X 10 ⁴ cells/well and incubated for 24h at 28 ℃. Mu.g of pEGFP-N3 plasmid was transfected with FISHTRANS transfection reagent (MeiSenTe Biotechnology, CHN) and FuGENE HD transfection reagent (Promega), respectively. After 48h of transfection, cells were washed 2 times with PBS and fixed with 4% Paraformaldehyde (PFA) for 1h. The fixative was removed and stained with 4', 6-diamino-2-phenylindole (DAPI) at a concentration of 1. Mu.g/mL in the dark at room temperature for 10min. Subsequently, the coverslip was washed to remove excess DAPI and placed on the slide. Stained cells on the coverslip were visualized using a Karl Zeiss upright fluorescence microscope (Axio imager M2) equipped with a 20-fold objective. Transfection efficiency was calculated by counting GFP positive cells and total number of cells in 10 independent light fields.

As shown in FIG. 6, after 48h of transfection, a clear green fluorescent signal was observed under a fluorescent microscope, indicating that the pEGFP-N3 plasmid was successfully transfected into the fin cells. About 70 cells showed GFP positivity out of about 220 cells per field of view when transfected with FISHTRANS transfection reagents. Therefore, the transfection efficiency of fin cells was about 32% (fig. 6A). While about 23 cells showed GFP positivity in about 90 cells per field of view when transfected with the FuGENE HD transfection reagent (fig. 6B). In addition, fuGENE HD transfection reagent had a damaging effect on fin cells, which were significantly less numerous than when transfected with FISHTRANS. In conclusion, the fin cell can be used for in vitro expression research of the exogenous gene by transfecting the exogenous expression plasmid through FISHTRANS more efficiently.

7. Salchinotus brothers fin cell expression of immune-related genes after polyI:C stimulation

To investigate the feasibility of fin cell lines for immunological studies, we evaluated the expression of immune-related genes in poly (I: C) -treated fin cells. The 55 th generation fin cells were inoculated into 6-well plates, incubated with 20% FBS medium for 24 hours, and when the degree of fusion reached 90%, 1. Mu.g/mL poly (I: C) was transfected into the cells, and after waiting for 0, 6, 12, 24 and 48 hours, cellular RNA samples were collected. Total RNA from cells was extracted according to the reagent instructions (Invitrogen). The RNA quality was determined by agarose gel electrophoresis. The purity and yield of RNA were determined using a spectrophotometer (Nanodrop 2000, thermo). First strand cDNA was synthesized using GoScript reverse transcription system (Promega) according to the manufacturer's instructions. qPCR was performed on CFX96 real-time system (BioRad) using Fast SYBR Green master mix (BioRad). The PCR conditions were: 95℃for 5min,95℃for 20s,60℃for 20s,72℃for 20s,40 cycles. The expression of the beta-actin of the pomfret brucei is used as an internal reference for normalization. The specificity of the PCR amplification of each primer set was analyzed by a dissolution profile. The relative gene expression level was calculated by the 2 ^-△△CT method.

The quantitative results show (see FIG. 7) that the expression levels of IFN system-related genes mita, irf3, irf7 in fin cells were significantly increased when poly (I: C) was stimulated for 12 h. The IFN gene up-regulates expression by 12 times, and the expression levels of IFN stimulating genes vig1 and isg are also significantly up-regulated. After 24h and 48h of poly (I: C) stimulation, the expression levels of the mita, irf7, ifn, vig1, isg genes were decreased. Indicating that the fin cells were stimulated with poly (I: C) for 12h, the intracellular immune response level was strongest, followed by a decrease in immune response level. The influence of Poly (I: C) on the expression of the innate immunity genes after infecting fin cells shows that the fin cell line of the trachinotus brothers is a useful tool for researching fish virus diseases and host reactions, and can be used for fish virus immunity research and detection.

It should be understood that the foregoing examples of the present invention are merely illustrative of the present invention and not limiting of the embodiments of the present invention, and that various other changes and modifications can be made by those skilled in the art based on the above description, and it is not intended to be exhaustive of all the embodiments of the present invention, and all obvious changes and modifications that come within the scope of the invention are defined by the following claims.

Claims (8)

1. A trachinotus brothers fin cell line with a preservation number of CCTCC NO: C2023386.

2. The method for constructing a trachinotus brothers fin cell line according to claim 1, comprising:

(1) Treatment of fin structure: cleaning fin rays of young pomfret of the trachinotus brucei by using an L-15 basal medium, shearing the young pomfret into pieces in the L-15 basal medium, cleaning the small pomfret by using a cell screen, inoculating the small pomfret to the bottom of a cell culture bottle, and slowly adding an L-15 complete medium to the other side of the cell culture bottle for culture;

(2) Primary culture: fresh L-15 complete medium was changed every 2-3 days during primary culture, and cells were removed from the tissue mass on day 7 of primary culture;

(3) Subculture: after the confluence of the fin cells of the pompano reaches 90%, sucking out the old culture medium, adding PBS for cleaning once, digesting the adherent cells for about 2min by using 0.25% pancreatin containing EDTA, observing the cells under an inverted microscope to round and start falling off, adding a complete cell culture medium to stop digestion, lightly blowing and dispersing the suspended cells, and carrying out cell splitting and subculturing.

3. The construction method according to claim 2, wherein the L-15 basal medium contains 400U/mL of a mixture of green streptomycin, and the L-15 complete medium contains 20% fetal bovine serum, 400U/mL of a mixture of green streptomycin, and 0.01% recombinant human basic fibroblast growth factor.

4. The construction method according to claim 2, wherein in the step (1), the culture flask is placed in an incubator at 28℃for inversion culture, and after the dry application for 6 hours, the flask is slowly turned over and placed in the incubator.

5. The method according to claim 2, wherein in the step (3), the cells are bottled at a ratio of 1:2 and placed in a 28℃incubator for subculture.

6. The use of a trachinotus brothers fin cell line according to claim 1 for immunodetection of expression of exogenous genes and viral stimulators.

7. The use according to claim 6, wherein the trachinotus brothers fin cells are efficiently transfected with exogenous expression plasmid by FISHTRANS.

8. The use according to claim 6, wherein the expression levels of IFN system-related genes mita, irf3, irf, IFN, vig1 and isg in fin cells of pompano are markedly increased when the fin cells are stimulated with poly (I: C) for 12 h.