CN114058566B - Mandarin skin cell line and application thereof - Google Patents

Abstract

The invention relates to a mandarin fish skin cell line SCS and its application. The cell line was preserved in the China Center for Type Culture Collection (CCTCC) on July 22, 2021, and the preservation number is: CCTCC NO: C2021198. SCS is composed of cells with fiber-like morphology. The karyotype is 2n=48, accounting for 44% of the number of chromosomes in the observed division phase. The karyotype formula is 2n=1m+1sm+18t+4st, and the doubling time of passage is 3 days. Aquatic animal viruses are susceptible and can be genetically manipulated. The present invention is the first time in the field to obtain a mandarin skin cell line, which can be used to express foreign proteins, isolate and cultivate aquatic animal viruses, and can also be used as a monitoring index for aquatic animal viruses in water bodies, and can be used to detect whether aquatic animals are virus infection.

Description

A kind of mandarin fish skin cell line and its application

technical field

The invention relates to the field of aquatic animal cells, and more particularly relates to a mandarin fish skin cell line and its application.

Background technique

Siniperca chuatsi (Siniperca chuatsi) belongs to Perciformes, Siniperidae, and Siniperca genus. It is a characteristic economic fish in my country. It is popular because of its delicious meat, no intermuscular spines, and rich nutrition. Its breeding scale and output are increasing day by day, but its large-scale farming is often plagued by diseases, especially diseases caused by viral pathogens.

Since viruses need to proliferate in cells, cell culture technology plays an important role in the detection and identification of virus pathogens, the determination of physical and chemical properties, antigen preparation and vaccine production, research on the interaction between pathogens and hosts, and epidemiological investigation and prevention. Due to the special living environment, skin tissue is an important physical and physiological barrier for fish to resist pathogen invasion, but cultured cell lines derived from fish skin tissue are rare. The skin tissue culture cell line of mandarin fish has not been reported yet.

Contents of the invention

The inventor created a mandarin fish skin cell line SCS in his work, which was deposited in the China Center for Type Culture Collection (CCTCC) on July 22, 2021, with the preservation number: CCTCC NO: C2021198. SCS is composed of cells with fiber-like morphology. The karyotype is 2n=48, which accounts for 44% of the number of chromosomes in the observed cleavage phase. The karyotype formula is 2n=1m+1sm+18t+4st. Animal viruses Siniperca chuatsi rhabdovirus (SCRV), giant salamander frog virus (Andrias davidianus ranavirus, ADRV), swamp green bullfrog virus (Rana grylio virus, RGV), turbot rhabdovirus (Scophthalmus maximus rhabdovirus, SMRV) , Paralichthys olivaceus rhabdovirus (PORV) and grass carp reovirus (GCRV) are susceptible to various aquatic animal viruses and can be genetically manipulated.

The present invention also provides the application of the above-mentioned mandarin fish skin cell line in protein expression. Since SCS can be genetically manipulated, and foreign genes can be effectively expressed in cells, SCS can be used for protein expression, production of protein products, preparation of vaccines, etc.

Because SCS is susceptible to a variety of aquatic animal viruses, the present invention also provides the application of the above-mentioned mandarin fish skin cell line in the isolation or cultivation of aquatic animal viruses, which can be isolated from water bodies or infected animals or cultivate these susceptible viruses, providing The corresponding biological products meet the needs of production or research.

In addition, the present invention also provides the application of the above-mentioned mandarin fish skin cell line in detecting aquatic animal viruses in water bodies.

The present invention also provides a method for detecting aquatic animal viruses, comprising the following steps:

S1: After the water sample to be tested is filtered and sterilized or the animal tissue is homogenized, it is added to the culture environment of the above-mentioned mandarin fish skin cell line;

S2: Observing the characteristic changes of the mandarin fish skin cell line cultured in S1.

In a specific embodiment, said characteristic changes include changes in cell morphology and/or changes in gene expression. After 24 hours of inoculation, the cell morphology of SCS can undergo obvious changes. Therefore, the change of cell morphology can be used as one of the evaluation indicators for the presence of aquatic animal viruses in the sample to be tested.

After 3 hours of inoculation, the expression of some immune-related genes in SCS was different. Therefore, the expression changes of specific immune genes can be used as one of the evaluation indicators for the presence of aquatic animal viruses in the samples to be tested.

In a specific embodiment, said changes in gene expression comprise changes in the expression of one or more combinations of CD3, IRF1, LGP2 and IL8.

Deposit of Cell Lines

The mandarin fish skin cell line mentioned in the present invention has been preserved in the Chinese Typical Culture Collection Center (CCTCC) of Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province on July 22, 2021, and the preservation number is: CCTCC NO : C2021198, taxonomically named mandarin fish skin cell line SCS.

Description of drawings

Fig. 1 is a photomicrograph of SCS of mandarin fish skin cell line. Bar = 50 μm.

Fig. 2 is the growth curve of SCS of mandarin fish skin cell line.

Figure 3 is the karyotype of the SCS line of mandarin fish skin cells. Among them, A is the chromosome statistical distribution map of 150 cells; B is the chromosome karyotype map of representative cells.

Fig. 4 is a photomicrograph of the SCS skin cell line of mandarin fish inoculated with different aquatic animal viruses. Bar = 100 μm.

Fig. 5 is a fluorescence micrograph of the GFP gene transferred into the mandarin fish skin cell line SCS. GFP is green fluorescent protein.

Fig. 6 is a graph showing the expression statistics of immune-related genes in the mandarin fish skin cell line SCS after inoculation.

Detailed ways

The principles and features of the present invention are described below in conjunction with the accompanying drawings, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

1. Construction of mandarin fish skin cell line SCS

Take a healthy mandarin fish (about 500g) for primary culture of skin cells. The specific operation is: put the fish to death, and put it in 75% alcohol for disinfection. Then the skin on both sides of the back was carefully peeled off, and placed in the M199 medium containing penicillin-streptomycin double antibody (1000 units/ml) for 3 hours. The soaked skin tissue was cut into pieces of about 1 mm ³ with sterile scissors, and then transferred to cell culture flasks. Invert the cell culture flask for about 4 hours, then add M199 medium containing 20% calf serum and double penicillin-streptomycin (100 units/ml), and place in a 25°C incubator for cultivation. Change half of the medium every 3-4 days until the cell monolayer grows out.

Subculture when the monolayer cells cover about 80% of the cell bottle: use 0.25% trypsin to digest the cells, passage at a ratio of 1:2, still use 20% calf serum and penicillin-streptomycin double antibody (100 units/ ml) of M199 medium. The digested and passaged cells were cultured in a 25°C incubator, and half of the medium was replaced every 3-4 days. After the cells were congested, the cells were still passaged in this way.

After repeated subculture and screening, a stable subcultured mandarin fish skin cell line SCS was obtained. The cell morphology of SCS is shown in Figure 1, which is a fibrous morphology, which is significantly different from the existing cell lines derived from mandarin fish tissue.

2. Preservation of SCS cell lines

The obtained SCS cell line was deposited in the Chinese Type Culture Collection Center (CCTCC) of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province on July 22, 2021, and the preservation number is: CCTCC NO: C2021198.

3. SCS culture and growth curve determination

SCS was digested and passed to multiple ^25cm2 cell culture flasks, so that the number of cells covered about 25% of the bottom area of the cell flasks, cultured at 25°C, three flasks were taken out for digestion every day, and the cell density and number were measured.

The results are shown in FIG. 2 , the cell density reached 2 times of the initial density 3 days after the SCS cells were subcultured, that is, the doubling time was 3 days.

4. SCS karyotype analysis

Passage SCS skin cells of mandarin fish into 25 cm ² cell culture flasks and place them in an incubator at 25°C. After 2 days of culture, colchicine was added at a final concentration of 2 μg/mL. After 10 hours of treatment, the cells were trypsinized, collected, and resuspended in 0.075M KCl. After 30 min, Carnoy's fixative solution (methanol: acetic acid = 3:1) was added and left at room temperature for 5 min. Cells were collected by centrifugation, resuspended in 5 mL of fresh Carnoy's fixative, and placed overnight at 4°C. Cells left overnight were collected by centrifugation and resuspended in 0.5 mL of fixative. Take an appropriate amount of the resuspension, drop it onto a pre-cooled glass slide, and stain it with Giemsa stain after drying. Observe and take pictures under an inverted microscope.

As shown in Figure 3, the karyotype of SCS in mandarin fish skin cells was 2n=48, which accounted for 44% of the number of observed chromosomes in cleavage phase (Figure 3A). It contains 1 pair of centrocentric chromosomes (m), 1 pair of subcentrocentric chromosomes (sm), 18 pairs of telocentric chromosomes (t) and 4 pairs of subtelocentric chromosomes (st) (Figure 3B ), the karyotype formula is: 2n=1m+1sm+18t+4st.

Compared with the other two cell lines derived from mandarin fish tissue, MFF-1 (mandarin mandarin embryo cell line) cells have three main distributions of chromosome numbers: 32, 36, and 48, among which the number of cells with 32 chromosomes is the largest; Brain cell line) cells have three major distributions of chromosome numbers: 36, 48, and 54, among which the number of cells with 36 chromosomes is the largest. SCS cells had the most number of 48 chromosomes. Therefore, karyotype analysis proved that SCS cells were significantly different from the other two mandarin mandarin tissue-derived cell lines.

5. Determination of the sensitivity of SCS to aquatic animal viruses

Three aquatic animal viruses, Siniperca chuatsi rhabdovirus (SCRV), giant salamander frog virus (Andrias davidianus ranavirus, ADRV) and swamp green bullfrog virus (Rana gryliovirus, RGV) were used for virus susceptibility testing. Passage the SCS to a 25 cm ² cell culture flask and place it in a 25°C incubator until it grows to a monolayer. The three viruses were inoculated into SCS cells at a dose of 0.1 MOI respectively. The infected cells continued to be cultured in a 25°C incubator, and were photographed at different time points for observation.

As shown in Figure 4, the three viruses (SCRV, ADRV and RGV) can all cause cytopathic changes in SCS cells, including cell shrinkage, cell lysis and shedding, irregular cavities in monolayer cultured cells, etc., and the cytotoxicity of the viruses The lesion effect was very obvious 24 hours after virus inoculation, indicating that SCS cells were very sensitive to these three viruses.

We also conducted corresponding experiments on Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PORV) and grass carp reovirus (GCRV), and observed to the susceptibility of SCS to these viruses and exhibit changes in morphology and gene expression following infection. It can be seen that SCS is broadly susceptible to a variety of aquatic animal viruses and produces an infectious phenotype.

This experiment shows that SCS can be used to inoculate and propagate viruses in aquatic animals. In addition, due to the susceptibility of SCS to aquatic animal viruses, it can show very obvious disease symptoms 24 hours after inoculation, so it can be used for water environment monitoring, especially in freshwater fish breeding waters, to detect whether there are For example, fish such as mandarin fish are susceptible to aquatic animal viruses, or to detect whether aquatic animals carry viruses.

6. Expression test of exogenous gene in SCS

The exogenous gene coding plasmid was transfected into SCS skin cells of mandarin fish, and its expression was observed. The specific steps are as follows: firstly, subculture the SCS cells into 24-well plates, place them in an incubator at 25° C., and perform exogenous gene transfection when the cell density is about 80-90%. Add 0.5 μg of the plasmid encoding green fluorescent protein (exogenous gene encoding plasmid) and 2 μl of transfection reagent (lipofectamine 3000) to 50 μl of serum-free medium, mix well, and place at room temperature for 20 minutes. Then it was added to the SCS cells in the 24-well plate, cultured at 25°C, observed and photographed under a fluorescent microscope after 48 hours.

As shown in Figure 5, 48 hours after transfection, some SCS cells could be observed to emit green fluorescence, and the green fluorescence was distributed throughout the cells. This shows that the SCS cell line we constructed can be conveniently genetically manipulated to express foreign genes in the cells. Therefore, if necessary, the gene expression cassette can be transferred into SCS to express the target protein.

7. Expression analysis of immune-related genes in SCS during virus infection

Two viruses, SCRV and ADRV, were used to infect the SCS of mandarin fish skin cells, and the expression of immune-related genes in mandarin fish was detected. The specific steps are as follows: subculture the SCS into a 25 cm ² cell culture flask, and culture it at 25° C. until the monolayer is covered. Connect to SCRV and ADRV respectively. Cells were collected at 0, 3, 6, and 12 hours after inoculation, and total RNA was extracted using Trizol reagent (Invitrogen). Reverse transcription was performed using HiScript III RT SuperMix for qPCR (+gDNA wiper) reagents (Vazyme). Finally, gene expression was detected by fluorescent quantitative PCR.

Among them, for ADRV-infected cells, the detected genes are CD3 (signal transduction molecule on the surface of T cells) and IRF1 (interferon regulatory factor 1, an important molecule in antiviral innate immunity); for SCRV-infected cells, the detected gene is LGP2 (RLR family molecule, an important pattern recognition receptor for the body to respond to RNA virus infection) and IL8 (interleukin-8, an important signaling molecule for inflammatory response).

As shown in Figure 6, ADRV infection caused a significant up-regulation of CD3 and IRF1. Among the time points tested, the up-regulation was most obvious at 12 h after infection, and the up-regulation of CD3 was larger; SCRV infection caused a rapid up-regulation of LGP2, and the up-regulation of At 6h, the expression level of LGP2 was up-regulated several times, while the expression level of IL-8 showed a trend of first decreasing and then increasing after SCRV infection.

By detecting the expression of related genes, it can be roughly determined whether there are fish-susceptible viruses in the water body, and the type of viruses.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (7)

1. A mandarin fish skin cell line, characterized in that it was preserved in the China Center for Type Culture Collection on July 22, 2021, and the preservation number is: CCTCC NO: C2021198. 2. the application of the mandarin fish skin cell line described in claim 1 in protein expression. 3. the application of the mandarin fish skin cell line described in claim 1 in separating or cultivating aquatic animal virus. 4. the application of the mandarin fish skin cell line described in claim 1 in the detection of aquatic animal virus. 5. A method for detecting aquatic animal virus, is characterized in that, comprises the following steps: S1: adding the sample to be detected to the culture environment of the mandarin fish skin cell line described in claim 1; S2: Observing the characteristic changes of the mandarin fish skin cell line cultured in S1. 6. The method according to claim 5, wherein the characteristic changes include changes in cell morphology and/or gene expression. 7 . The method according to claim 6 , wherein the gene expression changes include expression changes of one or more combinations of CD3, IRF1 , LGP2 and IL8.

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