Insect ovarian tissue cell line sfo-25 for culturing tetrapod iridovirus and construction method of insect ovarian tissue cell line sfo-25

Abstract

The invention discloses an insect ovarian tissue cell line sfo-25 for culturing a decapod iridovirus and a construction method of the insect ovarian tissue cell line sfo-25, and particularly relates to the field of aquatic organism cells and aquaculture disease prevention and control, and the insect ovarian tissue cell line sfo-25 is obtained by subculturing a spodoptera frugiperda ovarian tissue cell line. The construction method of the insect ovarian tissue cell line sfo-25 comprises the following steps: taking a spodoptera frugiperda ovarian tissue cell line for resuscitation, carrying out subculture by using a No.1 culture solution until the cell line is stable to obtain a stable insect ovarian tissue cell line, and carrying out domestication culture and subculture to obtain the insect ovarian tissue cell line sfo-25. The insect ovarian tissue cell line sfo-25 has the advantages of fast growth, low cost and sensitivity to full-eye iridovirus.

Description

Technical Field

[0001] The present application relates to the field of aquatic biological cells and aquaculture disease prevention and control, and in particular to an insect ovarian tissue cell line SFO-25 for culturing decapod iridescent virus and a construction method thereof. Background Art

[0002] Macrobrachium rosenbergii Macrobrachium rosenbergii ) is one of the largest freshwater shrimps in the world, native to the entire South Asia and Southeast Asia, as well as the northern Oceania and the western Pacific islands. Since the introduction of Macrobrachium rosenbergii from Japan in 1976, after more than 40 years of development and improvement, my country's Macrobrachium rosenbergii aquaculture industry has grown from scratch and has become an important industry for fishermen to increase their income and become rich. However, with the continuous expansion of aquaculture scale and the deterioration of aquaculture environment, diseases have become a major bottleneck restricting the development of these species industries, especially the decapod iridovirus disease, which has the characteristics of rapid onset, high mortality, wide range of species damage, and rapid spread, causing huge economic losses to Macrobrachium rosenbergii aquaculture. In 2019, the International Committee on Taxonomy of Viruses classified the shrimp hemocyte iridovirus that harms Penaeus vannamei, Macrobrachium rosenbergii, etc. and the red claw crayfish iridovirus derived from red claw crayfish into the genus Decapod iridescent virus, named Decapod iridescent virus 1 (DIV1). DIV1 is mainly prevalent in May and June and can infect shrimp of all sizes. The detection rate of the virus in shrimp with a body length of 4 to 7 cm is the highest. After being infected with DIV1, the shrimps move slowly and their vitality drops sharply, showing obvious external symptoms, mainly including weak shrimps, visible white triangles under the cephalothorax (often called "white heads" or "white spots"), empty stomach and jejunum, soft shrimp shells, red and even broken intestines, and pale and atrophic liver and pancreas. The virus has a fast transmission rate and a wide host range. It mainly breaks out in coastal breeding areas such as Zhejiang, Guangdong and Jiangsu. The virus detection rate and mortality rate of diseased shrimps are high. It has caused significant economic losses to the Macrobrachium rosenbergii breeding industry and seriously threatened the healthy development of the industry. To this end, Chinese invention patent CN202210749728.9 discloses a heat shock treatment method for reducing or purifying decapod virus 1 infected in shrimps and crabs for non-therapeutic purposes. The activity of the DIV1 virus is reduced by a heat shock method at 36°C, thereby achieving the purpose of treatment. At the same time, Chinese invention patent CN202211367931.6 discloses a visual rapid detection method for decapod iridescent virus 1 based on ERA technology and detection primers and probes for specific detection of DIV1 virus.

[0003] For researchers, the understanding of the above viruses, the analysis of the pathogenesis of the disease and drug screening are still achieved through live animal experiments. And the above live animal experiments have the following defects: 1. Live animal experiments require large breeding sites and equipment, which are affected by the seasons and waste a lot of costs and time; 2. Live animal experiments are not easy to control experimental conditions and are affected by the external temperature, dissolved oxygen content and animal diseases.

[0004] At the same time, Chinese invention patent CN202310578264.4 discloses a mandarin fish spinal cord tissue cell line-37 and its construction method and application. By constructing mandarin fish spinal cord tissue cell line-37, it is applied to frog iris virus, mandarin fish infectious spleen and kidney necrosis virus, and rhabdovirus culture. Although the above patent discloses the construction of cell lines and realizes the study of the above viruses from the cell culture level, it is only for fish. At present, there is no stable cell line construction in shrimp virus culture. The in vitro cell culture of decapod iris virus is still a major problem restricting the study of Macrobrachium rosenbergii diseases. Summary of the invention

[0005] The invention provides an insect ovary tissue cell line SFO-25 for culturing decapod iridescent virus. The insect ovary tissue cell line SFO-25 has the advantages of fast growth, low cost and sensitivity to decapod iridescent virus.

[0006] The objective of the present invention is achieved through the following technical solutions: An insect ovarian tissue cell line SFO-25 for culturing decapod iridescent virus, characterized in that it is obtained by subculture of the ovarian tissue cell line of Spodoptera frugiperda.

[0007] As a preference, the method comprises the following steps: (1) Passaging of insect ovarian tissue cell lines: The fall armyworm ovarian tissue cell line was revived and subcultured using No. 1 culture medium until it was stable, thereby obtaining an insect ovarian tissue cell line after stable subculture, which was recorded as sfo-1 cell line; (2) Domestication and cultivation: (2.1) The sfo-1 cell line was digested with trypsin-EDTA digestion solution to prepare a cell suspension, which was subcultured until stable to obtain the sfo-2 cell line; Wherein, in the above steps, the culture medium selected for subculture is culture medium No. 2; (2.2) The sfo-2 cell line was digested with trypsin-EDTA digestion solution to prepare a cell suspension, which was subcultured until stable to obtain the sfo-3 cell line; Wherein, in the above steps, the culture medium selected for subculture is culture medium No. 3; (2.3) The sfo-3 cell line was digested with trypsin-EDTA digestion solution to prepare a cell suspension, which was subcultured until stable to obtain the sfo-4 cell line; Wherein, in the above steps, the culture medium selected for subculture is No. 4 culture medium; (3) Subculture: The sfo-4 cell line was subcultured in medium No. 4 every 4-5 days to obtain the insect ovary tissue cell line-25.

[0008] Preferably, in step (1), culture medium No. 1 is an insect cell culture medium containing 100 IU / ml penicillin, 100 μg / ml streptomycin, and 15% fetal bovine serum.

[0009] Preferably, in step (2.1), the digestion step is: S01, aspirate the original culture medium into a centrifuge tube; S02, add 1 ml of 0.25% trypsin-EDTA digestion solution and digest at room temperature. After 3-5 minutes, remove trypsin-EDTA, add 1 ml of No. 2 culture medium to terminate digestion and blow the cells, collect the cell suspension, centrifuge it with the original culture medium for 5 minutes, and discard the supernatant after centrifugation; S03, add fresh culture medium, divide the bottles at a ratio of 1:2, place in a 28°C constant temperature incubator to start subculture, and replace No. 2 culture medium every 2-3 days; S04. When the confluence reaches 80-90%, use No. 2 culture medium for subculture until stable.

[0010] Preferably, the No. 2 culture medium is an insect cell culture medium containing 100 IU / ml penicillin, 100 μg / ml streptomycin, and 10% fetal bovine serum.

[0011] Preferably, in step S02, the centrifugal speed is 1000 rpm / min.

[0012] Preferably, the No. 3 culture medium is an insect cell culture medium containing 100 IU / ml penicillin, 100 μg / ml streptomycin, and 20% newborn calf serum.

[0013] Preferably, culture medium No. 4 is an insect cell culture medium containing 100 IU / ml penicillin, 100 μg / ml streptomycin, and 10% newborn calf serum.

[0014] The culture medium in No. 1 culture medium, No. 2 culture medium, No. 3 culture medium, and No. 4 culture medium all further include basic components, and the basic components include: Inorganic salts: including calcium chloride dihydrate, magnesium chloride hexahydrate, magnesium sulfate, potassium chloride; Amino acids: including L-tyrosine, L-valine, L-proline, L-arginine hydrochloride, D-fructose (F)-hexose, L-lysine hydrochloride, L-phenylalanine, L-threonine, L-serine; Vitamins: Biotin, Choline Chloride, Calcium D-Pantothenate.

[0015] Compared with the prior art, the advantages or beneficial effects of the technical solution of the present application include: 1. The cell line can be continuously and stably subcultured, so the insect ovarian tissue cell line sfo-25 can be mass-produced. The serum used is cheap, and it has a good growth state after subculture and can be frozen and revived.

[0016] 2. The cell line domesticated by the present invention is sensitive to decapod iridescent virus and has a faster pathological change time, and can be well used for in vitro culture of decapod iridescent virus.

[0017] 3. In this study, by optimizing the cell culture process, the domesticated insect ovarian tissue cell line SFO-25 has a faster growth rate and a lower culture cost. In addition, the characteristics of the cell line were analyzed and the sensitivity test to the decapod iridescent virus was conducted, laying the foundation for the study of the pathogenic mechanism of the decapod iridescent virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a microscopic image of cells not infected with DIVI; Figure 2 This is a microscopic image of cells 1 day after infection with DIVI; Figure 3 This is a microscopic image of cells 3 days after infection with DIVI; Figure 4 This is a microscopic image of cells 5 days after infection with DIVI; Figure 5 This is a microscopic image of cells 7 days after infection with DIVI; Figure 6 Electron micrographs of cells infected with DIVI; Figure 7 is the electron microscopic image of cells not infected with DIVI; Figure 8 is the PCR amplification curve of viral DNA; Fig. 9 This is a statistical graph showing the changes in viral load of inoculated cells over time. DETAILED DESCRIPTION

[0019] The following will describe the implementation methods of the present application in detail with reference to the accompanying drawings and embodiments, so that the implementation process of how the present application applies technical means to solve technical problems and achieve corresponding technical effects can be fully understood and implemented accordingly. The embodiments of the present application and the various features in the embodiments can be combined with each other without conflict, and the technical solutions formed are all within the protection scope of the present application.

[0020] It should be clear that the embodiments described below are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by technicians in the relevant field without making creative work are within the scope of protection of the present application. Example

[0021] The method for constructing an insect ovarian tissue cell line sfo-25 for culturing decapod iridescent virus comprises the following steps: The ovarian tissue cell line of Spodoptera frugiperda was obtained from the laboratory and frozen in a liquid nitrogen tank. It was revived and subcultured for 30 generations.

[0022] Passaging of insect ovarian tissue cell lines: The fall armyworm ovarian tissue cell lines frozen in liquid nitrogen were revived and subcultured using culture medium No. 1 (insect cell culture medium containing 100 IU / ml penicillin, 100 μg / ml streptomycin, and 15% fetal bovine serum) until stable.

[0023] (2) Acclimation and culture: Take the insect ovarian tissue cell line after stable subculture and digest it with trypsin-EDTA digestion solution. The specific steps of digestion are as follows: aspirate the original culture medium and put it into a centrifuge tube; add 1ml trypsin-EDTA (0.25% Trypsin-EDTA (1X)) digestion solution and digest at room temperature. After 3-5 minutes, remove the trypsin-EDTA, add 1ml No. 2 culture medium (insect cell culture medium with 100IU / ml penicillin, 100μg / ml streptomycin, and 10% fetal bovine serum) to stop digestion and blow the cells, collect the cell suspension, centrifuge it with the original culture medium at 1000rpm / min for 5 minutes, aspirate the supernatant and discard it; add fresh culture medium, divide it into bottles at a ratio of 1:2, place it in a 28℃ constant temperature incubator to start subculture, and replace No. 2 culture medium every 2-3 days. When the confluence reaches 80-90%, use No. 2 culture medium for subculture until stable.

[0024] (3) Acclimation culture: Take the cells in (2) after stable subculture and digest them with trypsin-EDTA digestion solution. The specific steps of digestion are as follows: aspirate the original culture medium and put it into a centrifuge tube; add 1ml trypsin-EDTA (0.25% Trypsin-EDTA (1X)) digestion solution and digest at room temperature. After 3-5 minutes, remove the trypsin-EDTA, add 1ml No. 3 culture medium (cell culture medium with 100IU / ml penicillin, 100μg / ml streptomycin, and 20% newborn calf serum) to stop digestion and blow the cells, collect the cell suspension, centrifuge it with the original culture medium at 1000rpm / min for 5 minutes, aspirate the supernatant and discard it; add No. 3 culture medium, divide it into bottles at a ratio of 1:2, place it in a 28℃ constant temperature incubator to start subculture, and replace No. 3 culture medium every 2-3 days. When the confluence reaches 80-90%, use No. 3 culture medium for subculture and subculture until stable.

[0025] (4) Acclimation culture: Take the cells in (3) after stable subculture and digest them with trypsin-EDTA digestion solution. The specific steps of digestion are as follows: aspirate the original culture medium into a centrifuge tube; add 1ml trypsin-EDTA (0.25% Trypsin-EDTA (1X)) digestion solution, digest at room temperature for 3-5 minutes, remove trypsin-EDTA, add 1ml No. 4 culture medium (insect cell culture medium with 100IU / ml penicillin, 100μg / ml streptomycin, and 10% newborn calf serum) to stop digestion and blow the cells, collect the cell suspension, centrifuge it with the original culture medium at 1000rpm / min for 5 minutes, aspirate the supernatant and discard it; add No. 4 culture medium, divide it into bottles at a ratio of 1:2, place it in a 28℃ constant temperature incubator to start subculture, and replace No. 4 culture medium every 2-3 days. When the confluence reaches 80-90%, use No. 4 culture medium for subculture and subculture until stable.

[0026] (5) Subculture: Subculture is performed when the cell confluence reaches 80-90%, and subculture is performed every 4-5 days. Specifically, the original culture medium is aspirated into a centrifuge tube; 1 ml of trypsin-EDTA digestion solution is added, and digestion is carried out at room temperature for 3-5 min. After digestion, 1 ml of No. 4 culture medium is used to terminate digestion and the cells are blown to collect the cell suspension; centrifuge together with the original culture medium at 1000 rpm / min for 5 minutes, aspirate and discard the supernatant; add 5 ml of No. 4 culture medium to each bottle, divide the bottles at a ratio of 1:2, and place them in a constant temperature incubator at 28°C to start subculture.

[0027] Referring to the cell growth state in Example 1, round-like cells are the main morphology, such as Figure 1 As shown; the cells can be stably subcultured. Currently, the cells have been subcultured for more than 30 generations and domesticated to obtain the insect ovary tissue cell line-25, named: insect ovary tissue cell line-25, namely sfo-25.

[0028] Example 2 (Freezing and resuscitation of SFO-25 cell line cells) 1) Take sfo-25 in the logarithmic growth phase, digest it with trypsin-EDTA digestion solution, collect the cell suspension, centrifuge at 1000rpm / min for 5 minutes, and discard the supernatant. Add 1ml of cell freezing solution (90% fetal bovine serum + 10% DMSO), resuspend, and transfer to a 1.8ml sterile cryotube; place the cryotube in a program cooling box, freeze it at -80℃ overnight, and place it in liquid nitrogen for long-term storage the next day; 2) To revive the cells frozen in 1), remove the cryovials from liquid nitrogen and place them in 37°C water. Shake briefly until thawed. Add 5 ml of medium No. 4 and transfer to a T25 cell flask. Culture in a 37°C incubator and change the medium every other day.

[0029] The cell recovery activity is 80%-90%. The recovered cells can adhere to the wall and grow normally, and can be stably subcultured. The cells are not affected before and after freezing.

[0030] Example 3 (Virus sensitivity test of sfo-25) 1) Take 30 generations of sfo-25; 2) Observation of cytopathic effect (CPE): After sfo-25 cells were subcultured to T25 cell flasks, the original culture medium was aspirated and the cells were rinsed with PBS. 800 ml of the corresponding virus solution was added to the cell flask, incubated at 24°C for 1 h, the virus solution was aspirated, and 5 ml of insect cell culture medium containing 100 IU / ml penicillin, 100 μg / ml streptomycin, and 2% newborn calf serum was added. The cells were cultured in a 24°C incubator and observed daily with an inverted microscope. The microscopic images of cells not infected with DIVI are shown in Figure 1 As shown. Cell microscopy images on days 1, 3, 5, and 7 after infection with DIVI are shown in Figure 2-Figure 5 As shown in the figure. The cell morphology change is a description of the light microscopic observation after virus infection. After virus infection, the cells show irregular morphology. After observation, the cells have obvious pathological effects after 1 day. They are sensitive to DIV1 virus, and the sensitivity is strong, and the pathological time is short.

[0031] like Figure 6 and Figure 7 As shown, compared with uninfected cells, insect cells infected with DIV1 showed cell membrane rupture, vacuoles in the cytoplasm, and swelling and rupture of organelles.

[0032] like Figure 8 and Fig. 9As shown, the virus stock solution was diluted 2 times and 3 times and then tried to be infected, and the viral load of the infected cells was detected on the 5th, 6th and 7th days after infection. The data showed that the viral load increased with the increase in the number of days of infection, indicating that the sfo-25 cell line prepared by the present invention is highly sensitive to decapod iridescent virus and is suitable for the cultivation of decapod iridescent virus, thereby helping scientific researchers to understand the virus, analyze the pathogenesis of the disease and screen drugs. Example

[0033] Among them, the preparation method of the ovarian cell line of fall armyworm pupae in Example 1 includes the following steps: taking a female fall armyworm pupa in the middle stage of development and soaking it in a 75% ethanol solution for 10 minutes for surface disinfection; dissecting the insect to remove the ovarian tissue, and washing it with physiological saline for 2-3 times, and then washing it with cell culture fluid (containing 100IU / ml penicillin, 100μg / ml streptomycin and 15% fetal bovine serum) for 1-2 times; then placing it in a cell culture bottle rinsed with the above cell culture fluid, adding 2ml of cell culture fluid, and placing it in a cell culture incubator at 28°C; adding 3ml of cell culture fluid after culturing for 36 hours, and continuing to culture; after the third day, a large number of single cells can be observed around the tissue expanding to the periphery; around the 18th day, the cells fill the entire culture bottle and start to be subcultured; around the 5th day of cell subculture, the cells fill the entire culture bottle, and the second subculture begins, until the 8th generation, the cell growth gradually stabilizes.

[0034] The fall armyworm pupa ovary cell line in Example 1 was selected from cells of passage 30.

Claims

1. An insect ovarian tissue cell line SFO-25 for culturing decapod iridescent virus, characterized in that: It is obtained by subculture of the ovarian tissue cell line of Spodoptera frugiperda.

2. A method for constructing an insect ovarian tissue cell line sfo-25 for culturing decapod iridescent virus, characterized in that: The steps include: (1) Passaging of insect ovarian tissue cell lines: The fall armyworm ovarian tissue cell line was revived and subcultured using No. 1 culture medium until it was stable, thereby obtaining an insect ovarian tissue cell line after stable subculture, which was recorded as sfo-1 cell line; (2) Domestication and cultivation: (2.1) The sfo-1 cell line was digested with trypsin-EDTA digestion solution to prepare a cell suspension, which was subcultured until stable to obtain the sfo-2 cell line; Wherein, in the above steps, the culture medium selected for subculture is culture medium No. 2; (2.2) The sfo-2 cell line was digested with trypsin-EDTA digestion solution to prepare a cell suspension, which was subcultured until stable to obtain the sfo-3 cell line; Wherein, in the above steps, the culture medium selected for subculture is culture medium No. 3; (2.3) The sfo-3 cell line was digested with trypsin-EDTA digestion solution to prepare a cell suspension, which was subcultured until stable to obtain the sfo-4 cell line; Wherein, in the above steps, the culture medium selected for subculture is No. 4 culture medium; (3) Subculture: The sfo-4 cell line was subcultured in medium No. 4 every 4-5 days to obtain the insect ovary tissue cell line-25.

3. The construction method according to claim 2, characterized in that: In step (1), culture medium No. 1 is an insect cell culture medium containing 100 IU / ml penicillin, 100 μg / ml streptomycin, and 15% fetal bovine serum.

4. The construction method according to claim 2, characterized in that: In step (2.1), the digestion step is: S01, aspirate the original culture medium into a centrifuge tube; S02, add 1 ml of 0.25% trypsin-EDTA digestion solution and digest at room temperature. After 3-5 minutes, remove trypsin-EDTA, add 1 ml of No. 2 culture medium to terminate digestion and blow the cells, collect the cell suspension, centrifuge it with the original culture medium for 5 minutes, and discard the supernatant after centrifugation; S03, add fresh culture medium, divide the bottles at a ratio of 1:2, place in a 28°C constant temperature incubator to start subculture, and replace No. 2 culture medium every 2-3 days; S04. When the confluence reaches 80-90%, use No. 2 culture medium for subculture until stable.

5. The construction method according to claim 2, characterized in that: Culture medium No. 2 is an insect cell culture medium containing 100 IU / ml penicillin, 100 μg / ml streptomycin, and 10% fetal bovine serum.

6. The construction method according to claim 4, characterized in that: In step S02, the centrifugal speed is 1000 rpm / min.

7. The construction method according to claim 2, characterized in that: Culture medium No. 3 is an insect cell culture medium containing 100 IU / ml penicillin, 100 μg / ml streptomycin, and 20% newborn calf serum.

8. The construction method according to claim 2, characterized in that: Culture medium No. 4 is an insect cell culture medium containing 100 IU / ml penicillin, 100 μg / ml streptomycin, and 10% newborn calf serum.

9. The construction method according to any one of claims 2 to 8, characterized in that: No. 1 culture fluid, No. 2 culture fluid, No. 3 culture fluid, and No. 4 culture fluid all further include basic components, and the basic components include: Inorganic salts: including calcium chloride dihydrate, magnesium chloride hexahydrate, magnesium sulfate, potassium chloride; Amino acids: including L-tyrosine, L-valine, L-proline, L-arginine hydrochloride, D-fructose (F)-hexose, L-lysine hydrochloride, L-phenylalanine, L-threonine, L-serine; Vitamins: Biotin, Choline Chloride, Calcium D-Pantothenate.

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