CN113249308B - Perch arterial ball cell line and application and culture method thereof - Google Patents
Perch arterial ball cell line and application and culture method thereof Download PDFInfo
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Abstract
The invention discloses a Perolabrax japonicus arterial ball cell line which is a Perolabrax japonicus arterial ball tissue cell separated from a Perolabrax japonicus arterial ball tissue, wherein the preservation number of the Perolabrax japonicus arterial ball cell line is CCTCC NO: C202194. The invention also discloses application of the lateolabrax japonicus arterial ball cell line. The lateolabrax japonicus arterial ball cell line provided by the invention has sensitivity to various fish viruses, and has cytopathic effect after virus inoculation, so that the lateolabrax japonicus arterial ball cell line can be directly applied to pathogen research; and the transfection efficiency is higher, and after the exogenous recombinant plasmid is transfected, active protein can be expressed, so that the method can be used for scientific research.
Description
Technical Field
The invention relates to a cell line, in particular to a Perch arterial ball cell line. Meanwhile, relates to the application and the culture method of the lateolabrax japonicus arterial ball cell line.
Technical Field
Lateolabrax japonicus belongs to Osteichthyes, Perciformes, Percoidei and Serranidae, belongs to fishes with wide temperature and wide salinity, has the characteristics of quick growth, large size, delicious meat quality, high economic benefit and the like, and is a main variety of the seawater cultured Lateolabrax japonicus in China. The lateolabrax japonicus is widely distributed in offshore and estuary regions of China, Japan and Korean peninsula, can be propagated in seawater, can survive and grow in seawater, brackish, fresh water or estuary regions, and can be suitable for various water areas. The artificial culture area of the lateolabrax japonicus in China is wider, except Taiwan (without statistics) and Shanghai, the lateolabrax japonicus is cultured in various coastal provinces and cities in China, and the quantity of real estate is higher mainly in Guangdong, Fujian, Zhejiang, Guangxi, Shandong and the like. The fish has the advantages of high growth speed, wide salinity adaptation, low temperature resistance, less diseases, more and less meat, fresh and tender meat, rich nutrition and the like, and becomes the main seawater economic fish in China.
Under the natural environment, the disease resistance of the lateolabrax japonicus is strong, but with the continuous improvement of the intensification degree and the gradual expansion of the breeding mode and the breeding density, the occurrence condition of the lateolabrax japonicus diseases is increased day by day, and the disease incidence is more and more serious, including bacterial diseases and virus diseases with more and more various types. Among them, various viruses have the greatest harm to young lateolabrax japonicus and breeding, such as nervous Necrosis Virus (VNN), Infectious spleen and Kidney Necrosis Virus (Infectious spleen and Kidney Necrosis Virus, ISKNV), Red sea bream iridovirus (RSIV), frog Virus (Rana grylio Virus, RGV), which can infect lateolabrax japonicus larvae, juveniles, and adult fish, with the mortality rate being up to 90% or more, and can be horizontally transmitted by water sources, baits, and also can be vertically transmitted by parent fish spawning.
The outbreak of viral diseases seriously restricts the rapid development and variety promotion of the lateolabrax japonicus breeding industry. The fish cell line has great significance in fish virology research, and is mainly used for scientific research such as virus separation and identification, virus pathogenesis and the like, virus sensitivity research, virus proliferation, epidemiology research and the like. In addition, the cell line is an ideal material for gene function analysis, and has great significance for researching the functions of the genes related to the immune regulation. The establishment of primary fish cell lines is a relatively mature technology at present, but it is relatively difficult to obtain a virus-sensitive cell line, and virus sensitivity research needs to be carried out on the primary cell lines to screen the virus-sensitive cell lines. Different virus sensitive cells have differences, the viruses with larger harm to the perch at present comprise iridovirus frogs, iridovirus cytomegaviruses, nervous necrosis viruses and the like, although the scleroderma fish cell line used commercially at present can also replicate perch source viruses, the sensitivity is low, the virus titer is not high due to the distant relationship between the cells and the perch, and the virus sensitive cells are not beneficial to virus separation, identification, scientific research, vaccine development and the like.
To date, there have been reports on several cell lines such as brain cell line, head kidney cell line, liver cell line, etc. of lateolabrax japonicus, but there has been no report on arterial bulbar cell line of lateolabrax japonicus.
Disclosure of Invention
One of the objectives of the present invention is to provide a Perch arterial globulin cell line.
Specifically, the lateolabrax japonicus arterial corpuscle cell line is lateolabrax japonicus arterial corpuscle tissue cells separated from lateolabrax japonicus arterial corpuscle tissue, and the preservation number of the lateolabrax japonicus arterial corpuscle cell line is CCTCC NO: C202194.
The lateolabrax japonicus arterial sphere cell line can be stably and continuously passaged for at least 40 generations.
The second purpose of the invention is to provide the application of the lateolabrax japonicus arterial globulin cell line in culture of lateolabrax japonicus virus and/or detection of non-diagnostic therapeutic purpose.
The third purpose of the invention is to provide the application of the lateolabrax japonicus arterial ball cell line as a host cell for researching aquatic animal viruses.
The fourth purpose of the invention is to provide the application of the lateolabrax japonicus arterial ball cell line in preparing a drug screening model for resisting aquatic animal viruses.
The fifth purpose of the invention is to provide the application of the lateolabrax japonicus arterial ball cell line in the separation of aquatic animal viruses. The aquatic animal Virus is Infectious Spleen and Kidney Necrosis Virus (ISKNV), nervous Necrosis Virus (VNN), frog Virus (RGV).
The virus is a weever-derived infectious spleen and kidney necrosis virus, a weever-derived frog iridovirus and a weever-derived nervous necrosis virus.
The sixth purpose of the invention is to provide the application of the lateolabrax japonicus arterial ball cell line in expressing foreign protein.
The culture method of the lateolabrax japonicus arterial ball cell line provided by the invention comprises the following steps: culturing in M199 or L15 medium containing 10-20% fetal calf serum at 26-28 deg.C.
The construction method of the Perolabrax japonicus arterial ball cell line provided by the invention comprises the following steps:
cutting the lateolabrax japonicus arterial corpuscle tissue, putting the tissue into pancreatin digestive juice to be digested to a cell mass, collecting the digested cells to perform primary culture and subculture, and finally obtaining the lateolabrax japonicus arterial corpuscle cell line by screening stability and different fish virus sensitivities. The lateolabrax japonicus glomus cell line is preserved in China Center for Type Culture Collection (CCTCC) at 12.5.2021, the preservation number is CCTCC NO: C202194, the cell line is classified and named as lateolabrax japonicus glomus cell line LMAB, the place is as follows: wuhan university in Wuhan, China.
The invention has the advantages that:
1. the lateolabrax japonicus arterial ball cell line provided by the invention is successfully cultured in vitro for the first time, and obviously grows in an extending way when cultured for 3 days; after 7 days, the culture bottles are fully paved, and the digested culture bottles have stronger reproductive capacity and present typical fibroblast shapes; the cells are continuously subcultured for 5 months and are transferred to 40 generations, and the morphology and growth characteristics of the fibroblasts are kept all the time.
2. The Perolabrax japonicus arterial globulin cell line provided by the invention has stable characteristics, can be passaged to 40 generations at present, can maintain a good growth state, and can be subjected to cryopreservation.
3. The lateolabrax japonicus arterial ball cell line provided by the invention has sensitivity to various fish viruses, and has cytopathic effect after virus inoculation, so that the lateolabrax japonicus arterial ball cell line can be directly applied to pathogen research.
4. The Perolabrax japonicus arterial globulin cell line provided by the invention has higher transfection efficiency, can express active protein after transfecting exogenous recombinant plasmids, and can be used for scientific research.
Drawings
FIG. 1 is a diagram showing the morphology of the cells of the Perolabrax japonicus arterial ball in example 1 of the present invention.
A, primary cells of an arterial ball of the lateolabrax japonicus; b, 15-generation weever arterial glomerular cells; c, 30-generation weever arterial glomerular cells; d, 40-generation weever arterial glomerulus cells.
FIG. 2 is a diagram showing the screening of the optimum culture conditions for the arterial ball cell line of Japanese sea Perch in example 2 of the present invention.
FIG. 3 shows the results of the sensitivity experiment CPE of the Perciformis arterial ball cell line of the present invention to different aquatic animal viruses in example 4.
A, comparison; b, ISKNV infection for 3 d; c, RGV infection 6 d; d, VNN infection 3D.
FIG. 4 shows the results of the electrophoresis of the sensitivity test of the Perolabrax japonicus arterial ball cell line to different aquatic animal viruses in example 4 of the present invention; lane M, DNA molecular weight standard; lane 1, ISKNV infected lateolabrax japonicus arterial sphere cells; lane 2, lateolabrax japonicus arterial blast cells not infected with ISKNV; lanes 3, 4, first and second expansion of NVV infected lateolabrax japonicus arterial blast cells; lanes 5, 6, first and second expansion of NVV-uninfected lateolabrax japonicus arterial cells; lane 7, lateolabrax japonicus arterial sphere cells infected with RGV; lane 8, Perch arterial spherocytes not infected with RGV.
FIG. 5 shows the result of the expression of exogenous gene GFP transfected by Perca fluviatilis arterial sphere cell line in example 5 of the present invention. A. B, performing blank control on the lateolabrax japonicus arterial corpuscle cells without transfection of exogenous genes; C. and D, transfecting GFP-transfected lateolabrax japonicus arterial spherical cells.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
EXAMPLE 1 obtaining of Perch arterial globulin cell line
1. Primary culture
About 10g of young lateolabrax japonicus is taken, and after anesthesia, the young lateolabrax japonicus is soaked in 75% alcohol for 30 s. Quickly dissecting, separating tissue block of artery and sacculus, cleaning the tissue block with DPBS (400U/mL double antibody) for 2-3 times, and removing blood; placing the tissue blocks in a 6-well plate, adding 1ml of DPBS and 2ml of L-15 culture medium, cutting the inclined cell plate, sucking the cut inclined cell plate into a 15ml centrifuge tube, and filling the L-15 culture medium to 5 ml. Centrifuging, removing supernatant, adding 5ml of 0.25% trypsin into the precipitate, digesting at 37 ℃, and observing cell dispersion under a microscope in the digestion process until most tissue blocks are digested and dispersed into cell clusters; the digest was filtered using a disposable 200 mesh cell sieve. Transferring the filtrate into a centrifuge tube, centrifuging, and discarding the supernatant; suspending cells by using a complete culture medium containing 20 percent of serum and 200U/mL of serum of double antibody, and transferring the cells into a culture bottle, wherein the total volume in the culture bottle is about 5 mL; after 1-3 days of culture, the cells were attached, the fresh complete medium was replaced, and culture was continued for 2-3 days to obtain primary cells that grew to 80-90% confluence (FIG. 1). Wherein, the complete medium components: 20% fetal bovine serum, 100U/mL penicillin, 100U/mL streptomycin, and a culture solution of GIBICO L-15 with a pH value of 7.2-7.6.
2. Subculturing
After primary lateolabrax japonicus arterial spherical cells are fully paved in a monolayer culture bottle, sucking out culture solution by using a serum pipette, adding PBS (phosphate buffer solution) for cleaning twice, adding 2mL of 0.25% pancreatin digestive juice, observing cell rounding under an inverted microscope and beginning to fall off, adding 2mL of complete culture medium, blowing and beating suspended cells, carrying out cell seed division and passage according to a ratio of 1:2, adding the complete culture solution to 5 mL/bottle, and putting the bottle into a culture box at 28 ℃ for passage culture; passaging is done once about 3-5 days.
Example 2 determination of optimal serum concentration of Perch arterial ball cell line
Preparing culture solution with FBS concentration of 10%, 15%, 20% and 25%, respectively, and adjusting Perch arterial corpuscle cell concentration to 6 × 104mL-1The cells were seeded into 6-well plates, 2.5mL of culture solution per well, and cultured at 28 ℃ using the above four serum concentrations. 3-well cells were collected from each experimental group every day, and after trypsinization, the cells were collected and counted, and continuously cultured and counted for 1 week, and a growth curve was drawn, and it was found that FBS concentration of 20% to 25% was suitable for the culture of Perolax japonicus arterial ball cell line (FIG. 2). The culture solution is a complete culture medium and comprises the following components: 20% fetal calf serum, 100U/mL penicillin and 100U/mL streptomycin, and the pH value is 7.2-7.6, GIBICO L-15 culture solution.
Example 3 cryopreservation and recovery of cells
1. Cryopreservation of cells
Collecting 1 bottle (25 cm) by trypsinization2) The cells of the lateolabrax japonicus in the logarithmic growth phase are collected at the bottom of a centrifuge tube by centrifugation at 1000rpm/s for 5min, the supernatant is sucked by a pipette, 1mL of cell cryopreservation solution (containing 90% FBS and 10% MDSO) is added to resuspend the cells, then the cells are placed in a cryopreservation tube, the cryopreservation tube is placed in a programmed cooling box for overnight at-80 ℃, and finally the cells are placed in liquid nitrogen for long-term storage.
2. Cell resuscitation
Taking out the freezing tube containing cells from liquid nitrogen, rapidly freezing in 37 deg.C water bath, thawing, centrifuging (1000rpm/s for 5min), removing supernatant, adding complete culture medium to suspend cells, inoculating the resuspended cells to 25cm2The cells were cultured at 28 ℃ in a cell culture flask. Cell passage is carried out after the generation cells adhere to the wall and are fully paved. Complete medium composition: 20% fetal bovine serum, 100U/mL penicillin, 100U/mL streptomycin, and a culture solution of GIBICO L-15 with a pH value of 7.2-7.6.
Example 4 sensitivity of Perch arterial globulin cell lines to three common Perch viruses
The method comprises the following steps of (1) carrying out passage on the lateolabrax japonicus arterial glomerulus cells in the logarithmic growth phase, fully and uniformly mixing the cells, subpackaging the cells into a cell culture bottle, sucking out culture solution in the culture bottle when the cells grow to 80% -90% of the bottom of the bottle which can be fully filled, washing the cells for 2 times by using PBS, and respectively inoculating the cells into 2mL 3 of common lateolabrax japonicus viruses: infectious spleen and Kidney Necrosis Virus (Infectious splenic Necrosis and Kidney Necrosis Virus, ISKNV), nervous Necrosis Virus (VNN), frog Virus (Rana grilio Virus, RGV), PBS was used as a blank control group, after 1h of Virus incubation, Virus solution was aspirated, 5mL of cell maintenance medium (complete medium containing 5% FBS) was added, placed in a 28 ℃ cell culture chamber, and cell growth was observed daily for cytopathic effect (CPE) status. When the cells have CPE, after the cells are shed by 50%, collecting cell suspension, extracting total DNA, carrying out PCR virus detection, and analyzing whether the virus is proliferated in the Perch arterial cells. The results are shown in FIGS. 3-4.
Example 5 expression of the exogenous fluorescent protein GFP in Perch arterial cells
The GFP fluorescent protein nucleic acid sequence is connected to pcDNA31-myc-his (-) plasmid by adopting a double-enzyme cutting method to construct a GFP-pcDNA31-myc-his (-) recombinant plasmid, the correct connection of the recombinant plasmid is confirmed by sequencing by a sanger method, and the recombinant plasmid is extracted by using an endotoxin-removing plasmid miniprep kit (manufacturer: magenta, product number: P1154-02, product batch: DIK 09-01). Selecting Lateolabrax japonicus arterial saccule in logarithmic growth phase, inoculating in 24-hole cell culture plate, regulating cell concentration to 6 × 104mL-1. After the cells are attached to the wall, the extracted recombinant plasmid is transfected into the Japanese perch artery ball cells by a Lipofectamine 3000 transfection reagent. After 24h, the luminescence of the fluorescent protein is observed under a fluorescence inverted microscope.
Claims (9)
1. A Perch arterial corpuscle cell line is a Perch arterial corpuscle tissue cell separated from Perch arterial corpuscle tissue, and the preservation number of the Perch arterial corpuscle cell line is CCTCC NO: C202194.
2. The lateolabrax japonicus tenocyte line of claim 1, which is stable for serial passage for at least 40 passages.
3. Use of the lateolabrax japonicus arterial sphere cell line of claim 1 for culture of lateolabrax japonicus virus and/or detection for non-diagnostic therapeutic purposes.
4. Use of the lateolabrax japonicus arterial blast cell line of claim 1 as a host cell for the study of aquatic animal viruses.
5. Use of the lateolabrax japonicus arterial blast cell line of claim 1 as a drug screening model for preparing anti-aquatic animal virus.
6. Use of the lateolabrax japonicus arterial sphere cell line of claim 1 in the isolation of aquatic animal viruses.
7. The use according to claim 4 or 5 or 6, wherein the aquatic animal virus is infectious spleen and kidney necrosis virus, nervous necrosis virus and frog virus.
8. Use of the arterial cell line of Lateolabrax japonicus according to claim 1 for expressing foreign proteins.
9. The method for culturing an arterial ball cell line of Japanese sea bass according to claim 1 or 2, wherein the cells are cultured in M199 or L15 medium containing 10-20% fetal bovine serum at 26-28 ℃.
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| CN115595297B (en) * | 2022-09-22 | 2023-10-13 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus muscle cell line, construction method and application |
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Inventor after: Qiu Lihua Inventor after: Yan Lulu Inventor after: Guo Jieyun Inventor after: Zhao Chao Inventor after: Wang Pengfei Inventor after: Fan Sigang Inventor before: Yan Lulu Inventor before: Qiu Lihua Inventor before: Guo Jieyun Inventor before: Zhao Chao Inventor before: Wang Pengfei Inventor before: Fan Sigang |