CN114058566A - Mandarin skin cell line and application thereof - Google Patents

Mandarin skin cell line and application thereof Download PDF

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CN114058566A
CN114058566A CN202110945773.7A CN202110945773A CN114058566A CN 114058566 A CN114058566 A CN 114058566A CN 202110945773 A CN202110945773 A CN 202110945773A CN 114058566 A CN114058566 A CN 114058566A
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柯飞
张奇亚
孟献雨
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Institute of Hydrobiology of CAS
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Abstract

The invention relates to a mandarin fish skin cell line SCS and application thereof, wherein the cell line is preserved in China Center for Type Culture Collection (CCTCC) at 7-month and 22-month in 2021, and the preservation number is as follows: CCTCC NO: C2021198. SCS consists of fiber-like morphology cells, karyotype is 2n 48, accounting for 44% of the observed number of mitotic chromosomes, karyotype formula is 2n 1m +1sm +18t +4st, passage doubling time is 3 days, susceptibility to various aquatic viruses, and genetic manipulation is possible. The invention provides a mandarin fish skin cell line in the field for the first time, which can be used for expressing exogenous proteins and separating and culturing aquatic animal viruses, can also be used as a monitoring index of the aquatic animal viruses in a water body, and is used for detecting whether aquatic animals are infected with the viruses or not.

Description

Mandarin skin cell line and application thereof
Technical Field
The invention relates to the field of aquatic animal cells, in particular to a mandarin fish skin cell line and application thereof.
Background
Siniperca chuatsi belongs to Perciformes, bass and mandarin, is a characteristic economic fish in China, and is popular due to the characteristics of delicious meat, no muscle prick, rich nutrition and the like. The culture scale and the yield of the strain are increasing day by day, but the scale culture of the strain is often troubled by diseases, particularly diseases caused by viral pathogens.
Because the virus needs to be proliferated in cells, the cell culture technology plays an important role in the aspects of virus pathogen detection and identification, physicochemical property determination, antigen preparation and vaccine production, pathogen and host interaction research, epidemiological investigation and control and the like. 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 mandarin fish skin tissue culture cell line has not been reported yet.
Disclosure of Invention
The inventor creates a mandarin fish skin cell line SCS which is preserved in China Center for Type Culture Collection (CCTCC) at 7-22 months in 2021 with the preservation number as follows: CCTCC NO: C2021198. SCS is composed of fiber-like cells, the karyotype is 2n ═ 48, which accounts for 44% of the number of observed split-phase chromosomes, the karyotype formula is 2n ═ 1m +1sm +18t +4st, the passage doubling time is 3 days, and various aquatic viruses such as mandarin fish rhabdovirus (SCRV), giant salamander frog virus (ADRV), Rana nigromaculata virus (RGV), turbot rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (GCRV), and Grass carp enterovirus (GCRV) are susceptible to genetic manipulation.
The invention also provides application of the mandarin skin cell line in protein expression. Since SCS can be genetically manipulated and a foreign gene can be efficiently expressed in cells, SCS can be used for protein expression, production of protein products, preparation of vaccines, and the like.
As SCS is susceptible to various aquatic animal viruses, the invention also provides the application of the mandarin fish skin cell line in the separation or culture of aquatic animal viruses, which can be separated or cultured from water or toxic animals to provide corresponding biological products to meet the production or research requirements.
In addition, the invention also provides application of the mandarin fish skin cell line in detecting aquatic animal viruses in water.
The invention also provides a method for detecting aquatic animal viruses, which comprises the following steps:
s1: filtering a water sample to be detected for sterilization or homogenizing animal tissues, and then adding the water sample to be detected into the culture environment of the mandarin fish skin cell line;
s2: characteristic changes of the mandarin fish skin cell line cultured at S1 were observed.
In a particular embodiment, the characteristic change comprises a change in cell morphology and/or a change in gene expression. After 24h of inoculation, the cell morphology of SCS can be obviously changed, so that the change of the cell morphology can be used as one of the evaluation indexes for judging whether aquatic animal viruses exist in the sample to be detected.
After 3h of virus inoculation, some immune related genes of SCS have expression difference, so that the expression change of specific immune gene genes can be used as one of evaluation indexes for judging whether aquatic animal viruses exist in a sample to be detected.
In a specific embodiment, the gene expression changes include expression changes in a combination of one or more of CD3, IRF1, LGP2, and IL 8.
Preservation of cell lines
The mandarin fish skin cell line provided by the invention is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university, No. eight No. 299 in Wuchang district, Wuhan city, Hubei province within 22 months at 2021, with the preservation number: CCTCC NO: C2021198, taxonomically named mandarin fish skin cell line SCS.
Drawings
Fig. 1 is a SCS micrograph of mandarin fish skin cell line. Bar is 50 μm.
FIG. 2 is a graph of SCS growth curve of the mandarin fish skin cell line.
FIG. 3 shows the SCS lineage karyotype of mandarin fish skin cells. Wherein A is the chromosome statistical distribution map of 150 cells; b is a karyotype chart of representative cells.
FIG. 4 is a photomicrograph of the mandarin fish skin cell line SCS after inoculation with different aquatic animal viruses. Bar is 100 μm.
FIG. 5 is a fluorescent micrograph of the GFP gene introduced into the SCS, a mandarin fish skin cell line. GFP is a green fluorescent protein.
FIG. 6 is a statistical chart of the expression of immune-related genes after SCS vaccination of the mandarin fish skin cell line.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
1. Construction of mandarin fish skin cell line SCS
A healthy mandarin fish tail (about 500g) was taken for primary culture of skin cells. The specific operation is as follows: the fish were sacrificed and sterilized in 75% alcohol. The skin was then carefully peeled off on both sides of the back and placed in M199 medium containing penicillin-streptomycin double antibody (1000 units/ml) for 3 hours. Cutting the soaked skin tissue with sterile scissors to about 1mm3And then transferred to a cell culture flask. The cell culture flask was inverted for about 4 hours, and then M199 medium containing 20% calf serum and the dual antibody to streptomycin (100 units/ml) was added and placed in an incubator at 25 ℃. Half of the medium was changed every 3-4 days until a cell monolayer grew.
Subculture was performed when the monolayer cells were approximately 80% confluent in the cell flask: cells were digested with 0.25% trypsin, passaged at a ratio of 1:2, and M199 medium containing 20% calf serum and the dual resistance to streptomycin (100 units/ml) was used. And culturing the digested and passaged cells in an incubator at 25 ℃, and replacing half of the culture medium every 3-4 days. After the cells are full, they are still passaged in this way.
After repeated passage and screening, the mandarin fish skin cell line SCS with stable passage is obtained. The cell morphology of SCS is shown in fig. 1, and is a fibrous morphology, which is significantly different from the conventional mandarin fish tissue-derived cell line.
2. Preservation of SCS cell lines
The obtained SCS cell line is preserved in China type culture Collection (CCTCC) of Wuhan university with eight routes 299 in Wuchang district, Wuhan City, Hubei province within 2021, 7 months and 22 days, and the preservation number is as follows: CCTCC NO: C2021198.
3. Culture and growth Curve determination of SCS
SCS was digested and passaged to multiple 25cm2The cell culture flask of (4) was cultured at 25 ℃ so that the number of cells was spread over about 25% of the bottom area of the cell flask, and three flasks were taken out daily for digestion to measure the cell density and number.
As a result, as shown in fig. 2, the cell density reached 2 times the initial density after passage of SCS cells for 3 days, i.e., the doubling time was 3 days.
4. SCS karyotyping
Passage of mandarin fish skin cell SCS to 25cm2The cell culture flask is placed in an incubator at 25 ℃. After 2 days of culture colchicine was added to a final concentration of 2. mu.g/mL. After 10 hours of treatment, the cells were trypsinized, harvested and resuspended in 0.075M KCl. After 30min, Carnoy's fixative (methanol: acetic acid ═ 3:1) was added and left at room temperature for 5 min. Cells were harvested by centrifugation, resuspended in 5mL fresh Carnoy's fixative and left overnight at 4 ℃. Cells that were left overnight were harvested by centrifugation and resuspended in 0.5mL of fixative. A suitable amount of the resuspension was taken, dropped onto a pre-cooled slide, dried and stained with Giemsa stain. Observed under an inverted microscope and photographed.
As shown in fig. 3, the karyotype of mandarin fish skin cells SCS was 2 n-48, accounting for 44% of the number of mitotic chromosomes observed (fig. 3A). It comprises 1 pair of central centromeric chromosomes (m), 1 pair of sub-central centromeric chromosomes (sm), 18 pairs of centromeric chromosomes (t) and 4 pairs of sub-centromeric chromosomes (st) (FIG. 3B), the karyotype formula is: 2n is 1m +1sm +18t +4 st.
Compared with the other two mandarin-tissue-derived cell lines, MFF-1 (mandarin-embryo cell line) cells have three major chromosome number distributions of 32, 36 and 48, with the maximum number of cells having 32 chromosomes; CPB (mandarin brain cell line) cells have three major chromosome number distributions of 36, 48, and 54, of which the number of cells having 36 chromosomes is the largest. The SCS cells had the most number of 48 chromosomes. Therefore, karyotyping demonstrated significant differences between SCS cells and the other two mandarin tissue-derived cell lines.
5. Determination of SCS susceptibility to aquatic animal viruses
Virus susceptibility assays were performed using 3 aquatic animal viruses, mandarin fish rhabdovirus (SCRV), Andrias Davidianus Ranavirus (ADRV), and Rana Grylio Virus (RGV). Passage of SCS to 25cm2The cell culture flask was placed in an incubator at 25 ℃ until it was full of monolayers. These 3 viruses were individually inoculated into SCS cells at a dose of 0.1 MOI. The inoculated cells were further cultured in an incubator at 25 ℃ and observed by photographing at various time points.
As shown in FIG. 4, 3 viruses (SCRV, ADRV and RGV) all caused cytopathic effects of SCS cells, including cell shrinkage, cell lysis and detachment, irregular cavities of monolayer culture cells, and the like, and the cytopathic effects of the viruses were very obvious 24h after virus inoculation, indicating that the SCS cells were very sensitive to the 3 viruses.
Corresponding experiments are also carried out on Scophthalmus Maximus Rhabdovirus (SMRV), Paralichthys Olivaceus Rhabdovirus (PORV) and Grass reovirus (GCRV), the susceptibility of SCS to the viruses is also observed, and the SCS shows changes in morphology and gene expression after infection. It can be seen that SCS is widely 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 aquatic animal viruses. In addition, SCS is susceptible to aquatic animal viruses, and can show very obvious pathological symptoms 24h after inoculation, so that the method can be used for water environment monitoring, particularly water environment monitoring of freshwater fish culture water areas, detecting whether aquatic animal viruses susceptible to fishes such as mandarin fish exist in the water body or detecting whether aquatic animals carry the viruses or not.
6. Expression test of exogenous genes in SCS
Exogenous gene coding plasmid is transfected into mandarin fish skin cell SCS, and the expression condition is observed. The method comprises the following specific steps: first, SCS cells are subcultured into a 24-well plate and placed in an incubator at 25 ℃, and exogenous gene transfection is carried out when the cell density is about 80-90%. 0.5. mu.g of plasmid (exogenous gene-encoding plasmid) encoding green fluorescent protein and 2. mu.l of transfection reagent (lipofectamine 3000) were added to 50. mu.l of serum-free medium, mixed well, and left at room temperature for 20 min. Then, the cells were added to SCS cells in a 24-well plate, still cultured at 25 ℃ and observed under a fluorescent microscope and photographed after 48 hours.
As shown in FIG. 5, at 48h after transfection, part of SCS cells was observed to emit green fluorescence, and the green fluorescence was distributed throughout the cells. This demonstrates that the SCS cell line we constructed can be easily genetically manipulated to express foreign genes in cells. Therefore, if necessary, the gene expression cassette can be transferred to SCS for expression of the target protein.
7. Analysis of expression of immune-related genes in SCS upon viral infection
Two viruses, namely SCRV and ADRV are used for respectively infecting mandarin fish skin cell SCS, and the expression condition of mandarin fish immune related genes is detected. The method comprises the following specific steps: passage of SCS to 25cm2The cells were cultured in a cell culture flask at 25 ℃ until they grew into a monolayer. And respectively accessing the SCRV and the ADRV. Cells were harvested at 0, 3, 6, and 12h after the inoculation, and total RNA was extracted using Trizol reagent (Invitrogen). Reverse transcription was performed using HiScript III RT Supermix for qPCR (+ gDNA wiper) reagent (Vazyme). And finally detecting the gene expression by fluorescence quantitative PCR.
Among them, the detection genes for ADRV-infected cells were CD3(T cell surface signaling molecule) and IRF1 (interferon regulatory factor 1, an important molecule in antiviral innate immunity); the genes tested for cells infected with SCRV were LGP2 (an RLR family molecule, an important pattern recognition receptor for the body in response to RNA viral infection) and IL8 (interleukin-8, an important signaling molecule for inflammatory responses).
As shown in figure 6, ADRV infection caused significant upregulation of CD3 and IRF1, with upregulation most evident 12h post infection in the time points examined, with greater fold upregulation of CD 3; infection with SCRV caused a rapid up-regulation of LGP2, with the expression level of LGP2 being up-regulated several-fold at 6h post-infection, whereas the expression level of IL-8 showed a tendency to decrease and then increase following SCRV infection.
Whether the water body contains the fish susceptible virus or not and the type of the virus can be roughly determined by detecting the expression condition of the related genes.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (7)

1. The mandarin fish skin cell line is preserved in China center for type culture Collection at 22 months 7 and 2021 with the preservation number: CCTCC NO: C2021198.
2. Use of the mandarin skin cell line of claim 1 for protein expression.
3. Use of the mandarin fish skin cell line of claim 1 for isolating or culturing aquatic animal viruses.
4. Use of the mandarin skin cell line of claim 1 for detecting aquatic animal viruses.
5. A method for detecting viruses in aquatic animals, comprising the steps of:
s1: adding a sample to be detected to a culture environment of the mandarin skin cell line of claim 1;
s2: characteristic changes of the mandarin fish skin cell line cultured at S1 were observed.
6. The method of claim 5, wherein the characteristic change comprises a change in cell morphology and/or a change in gene expression.
7. The method of claim 6, wherein said gene expression changes comprise expression changes in a combination of one or more of CD3, IRF1, LGP2, and IL 8.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115948334A (en) * 2022-09-01 2023-04-11 广东永顺生物制药股份有限公司 Mandarin fish brain cell clone cell strain and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103275925A (en) * 2013-05-27 2013-09-04 中国水产科学研究院珠江水产研究所 Construction method of mandarin fish brain cell system
CN112280740A (en) * 2020-10-27 2021-01-29 中国水产科学研究院珠江水产研究所 Gobiocypris rarus brain cell line and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103275925A (en) * 2013-05-27 2013-09-04 中国水产科学研究院珠江水产研究所 Construction method of mandarin fish brain cell system
CN112280740A (en) * 2020-10-27 2021-01-29 中国水产科学研究院珠江水产研究所 Gobiocypris rarus brain cell line and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MENG XIAN-YU: "Development and characterization of a skin cell line from Chinese perch (Siniperca chuatsi) and its application in aquatic animal viruses", 《J FISH DIS》 *
陈晓武;申亚伟;赵金良;吴明林;: "鳜胚胎细胞系的建立与应用", 水产学报 *
雷存科: "三种水生动物细胞系对两株蛙病毒敏感性的比较", 《水产学报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115948334A (en) * 2022-09-01 2023-04-11 广东永顺生物制药股份有限公司 Mandarin fish brain cell clone cell strain and application thereof
CN115948334B (en) * 2022-09-01 2023-09-29 广东永顺生物制药股份有限公司 Mandarin fish brain cell clone cell strain and application thereof

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