A method for in vitro culturing Myxosoma portunus

Abstract

The present invention belongs to the field of biomedical technology, relates to the cultivation of vaccine antigens, and specifically relates to a method for culturing Hematodinium perezi in vitro. The RK-13 cells are cultured to a monolayer of cells, and Hematodinium perezi spores are added to the monolayer of cells for in vitro culture of Hematodinium perezi; during the in vitro culture of Hematodinium perezi, the culture medium is changed at regular intervals, and each time a part of the old culture medium is discarded and an equal amount of fresh culture medium is supplemented. The method of the present invention can culture Hematodinium perezi in vitro, can reproduce in large quantities and be stably passaged, and can provide a useful research model for the infection and pathogenic mechanism of Hematodinium perezi and for the development of prevention and control strategies.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to the cultivation of vaccine antigens. Specifically, it relates to a method for culturing Ameson portunus in vitro. Background Art

[0002] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Ameson portunus n.sp. is a new species of the genus Ameson of microsporidia and is also an emerging pathogen of Portunus trituberculatus. Epidemiological investigation results show that wild Portunus trituberculatus shows a high infection rate, and the infection rate in some epidemic areas is as high as over 90%, seriously threatening the growth of Portunus trituberculatus. The infection characteristics of Ameson portunus are that the early infection symptoms are not obvious, and the infection symptoms often appear only in the later stage of cultivation. At this time, the diseased crabs generally have a very high parasite load. Due to the intracellular parasitic characteristics of microsporidia and their strong chitinous shell, there is no effective prevention and control measure so far. After the temperature drops, the diseased crabs die in large numbers or even the pond harvest fails. The non-diseased crabs lose their commercial value due to the muscle albinism caused by Ameson portunus infection, commonly known as "toothpaste crabs", ultimately resulting in huge economic losses and seriously threatening the healthy development of the Portunus trituberculatus aquaculture industry.

[0004] Although more and more reports on primary cultured cells obtained from various organ sources of marine crustaceans have emerged, so far, there has been no report on the successful establishment of a marine crustacean cell line. In the case of the increasingly serious microsporidia infection in the shrimp and crab aquaculture industry, the lack of a suitable cell line for in vitro culture of microsporidia is an important reason restricting the research on microsporidia diseases of crustaceans, and it also restricts the research and development of Ameson portunus vaccines. Summary of the Invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a method for culturing Ameson portunus in vitro. The method of the present invention can culture Ameson portunus in vitro, can multiply in large quantities and be stably passaged, and can provide a useful research model for the infection and pathogenesis mechanism of Ameson portunus and for the development of prevention and control strategies.

[0006] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0007] On the one hand, an application of RK-13 cells in the in vitro culture of Ameson portunus.

[0008] The present invention uses mammalian cell lines (human foreskin fibroblasts (HFF), rabbit kidney cells (RK-13)), fish cell lines (grass carp gonad cells (GCO), zebrafish cells (ZF4)), and insect cell lines (insect ovary cells (SF-9)) to infect with purified spores of Kudoa portunus at the optimal growth temperature of each cell, and it is found that infection and proliferation only occur in RK-13 cells and can be stably passaged.

[0009] On the other hand, a method for culturing Kudoa portunus in vitro is to culture RK-13 cells to a monolayer of cells, add spores of Kudoa portunus to the monolayer of cells, and perform in vitro culture of Kudoa portunus; during the in vitro culture of Kudoa portunus, the culture medium is changed at regular intervals, and each time a part of the old culture medium is discarded and an equal amount of fresh culture medium is supplemented.

[0010] The method of the present invention can cause a very high infection rate in RK-13 cells, and a large number of spores in the cells can mature.

[0011] In the context of the increasingly serious microsporidiosis in crustaceans such as shrimps and crabs and fish, the method of the present invention provides an idea for the in vitro culture of various aquatic microsporidia. Thus, in the third aspect, an application of the above method in the in vitro culture of aquatic microsporidia other than Kudoa portunus.

[0012] Through the above-mentioned related solutions, the present invention has the following advantages:

[0013] The method for culturing Kudoa portunus in vitro provided by the present invention enables Kudoa portunus to be cultured in an environment outside the host, is not restricted by seasons, and a large number of highly active spores of Kudoa portunus can be obtained at any time. The present invention uses cells as a culture carrier and can culture and preserve Kudoa portunus for a long time and stably under suitable conditions.

[0014] The present invention cultures Kudoa portunus in vitro in the complete absence of crustacean cell lines, providing a good research model for microsporidiosis of Portunus trituberculatus. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0016] Figure 1 It is a line graph of the number of mature spores and a bar graph of the total number of spores (the total number of spores in the spore maturation period and mature spores) within 1 to 15 days after RK-13 cells are infected with Kudoa portunus in the embodiment of the present invention; each time, 3 wells in a 24-well plate are counted, and the infection amount is 1×10 8 spores / well.

[0017] Figure 2 This is a line graph showing the number of mature spores and a bar graph showing the total number of spores (the total number of spores in the spore maturation stage and mature spores) purified from infected RK-13 cells in the embodiments of the present invention within 1 to 15 days after infection of RK-13 cells; each time, 3 wells in a 24-well plate were counted, and the infection dose was 7×10 5 spores / well.

[0018] Figure 3 These are pictures taken during the infection of RK-13 cells with Myxosoma portunus in the embodiments of the present invention on the 8th and 11th days. The left column shows pictures of intracellular mature spores stained with Direct Yellow 96 (the scale bar for the 8th day of infection is 10 μm (upper), and the scale bar for the 11th day of infection is 5 μm (lower)). The middle column shows pictures of cell nuclei stained with DAPI (the scale bar for the 8th day of infection is 10 μm (upper), and the scale bar for the 11th day of infection is 5 μm (lower)). The right column shows pictures taken under bright field (the scale bar for the 8th day of infection is 10 μm (upper), and the scale bar for the 11th day of infection is 5 μm (lower)).

[0019] Figure 4 This is a bright field photo of the proliferation of Myxosoma portunus in RK-13 cells passed to the 8th generation in the embodiments of the present invention. Detailed implementation manners

[0020] It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0021] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0022] Since it is difficult to culture Myxosoma portunus in vitro by current methods, in order to solve the above technical problems, the present invention proposes a method for culturing Myxosoma portunus in vitro.

[0023] A typical embodiment of the present invention provides an application of RK-13 cells in culturing Myxosoma portunus in vitro.

[0024] In some embodiments, RK-13 cells are used as infected cells to culture Myxosoma portunus in vitro.

[0025] Another embodiment of the present invention provides a method for in vitro culturing Hematodinium sp. of swimming crab. Culture RK-13 cells to a monolayer of cells, add Hematodinium sp. spores of swimming crab to the monolayer of cells, and perform in vitro culture of Hematodinium sp. of swimming crab; during the in vitro culture of Hematodinium sp. of swimming crab, change the culture medium at regular intervals. Each time, discard a part of the old culture medium and supplement an equal amount of fresh culture medium.

[0026] In some embodiments, the culture medium for culturing RK-13 cells to a monolayer of cells is DMEM medium containing fetal bovine serum and double antibodies. More specifically, the content of fetal bovine serum is 9-11%, and the content of double antibodies is 0.9-1.1%.

[0027] In some embodiments, the in vitro culture of Hematodinium sp. of swimming crab is carried out in a CO2 incubator at 36-38°C and 4-6%.

[0028] In some embodiments, the interval time for changing the culture medium is 2-3 days.

[0029] In some embodiments, when changing the culture medium, discard 60-70% of the old culture medium.

[0030] In some embodiments, the preparation method of Hematodinium sp. spores of swimming crab is as follows: kill the pathogens on the surface of diseased crabs, then collect the muscles of diseased crabs, break the collected muscles of diseased crabs to obtain a tissue suspension, then filter and centrifuge the tissue suspension, and resuspend the spore precipitate with PBS to obtain the spores. The diseased crabs described in the present invention are swimming crabs suffering from Hematodinium sp. disease of swimming crab. More specifically, rinse the surface of the diseased crabs with water and then place them in 70-80% alcohol for 20-40 min to kill the pathogens on the surface of the diseased crabs. More specifically, use a 27G needle to break the muscles of the collected diseased crabs.

[0031] In some embodiments, it includes the subculture of Hematodinium sp. of swimming crab.

[0032] More specifically, the subculture is as follows: culture Hematodinium sp. of swimming crab in vitro for more than 12 days, remove exogenous spores, lyse the cells to release the Hematodinium sp. spores inside the cells, and infect new RK-13 cells with the released Hematodinium sp. spores.

[0033] More specifically, culture Hematodinium sp. of swimming crab in vitro for more than 6 days, digest the cells, and then add new RK-13 cells for in vitro culture.

[0034] More specifically, the method of the present invention preferably has the following steps:

[0035] (1) Cryopreservation of *Myxosoma portuni*: After rinsing the surface of the diseased crabs with water, place them in 75% alcohol for 30 min to kill the surface pathogens; carefully collect the muscle of the diseased crabs in a laminar flow hood and mix it with the cryopreservation solution at a ratio of 1:1, then store it in a -80 °C refrigerator for later use.

[0036] (2) Purification of *Myxosoma portuni* spores: Purify *Myxosoma portuni* spores from the muscle tissue of diseased crabs. Collect the muscle of the diseased crabs and disrupt the tissue through a 27G needle. After filtering and centrifuging the tissue suspension, resuspend the spore precipitate with PBS and store it at 4 °C for later use.

[0037] (3) Culture of RK-13 cells for infection: Seed RK-13 cells at a density of 1×10 5 ~1.5×10 5 per well in a 24-well plate, and culture the cells with DMEM medium containing 10% fetal bovine serum and 1% double antibiotics until the cells form a monolayer for later use.

[0038] (4) In vitro culture of *Myxosoma portuni*: Adjust the spore concentration to 1×10 8 cells / mL with cell culture medium, and add it to the monolayer cells in step (3), adding 1 mL per well, and continue to culture in a 37 °C, 5% CO2 incubator.

[0039] (5) Maintenance of *Myxosoma portuni* infection: Change the medium every 2 - 3 days. Each time, discard 2 / 3 of the old medium and supplement an equal amount of fresh medium, and continue to culture until the spores mature or use them for subculture during this period.

[0040] The in vitro proliferation of *Myxosoma portuni* is characterized by counting the number of spores produced at different infection times and staining the mature spores in RK-13 cells with Direct Yellow 96 (an aqueous solution of 10 μg / mL).

[0041] The subculture verification of *Myxosoma portuni* is as follows: Collect mature *Myxosoma portuni* spores from RK-13 cells infected with *Myxosoma portuni* for more than 13 days and reinfect RK-13 cells.

[0042] The third embodiment of the present invention provides an application of the above method in the in vitro culture of aquatic microsporidia other than *Myxosoma portuni*.

[0043] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific examples and comparative examples.

[0044] In the following examples, conventional cell biology methods are mainly used, and these methods are well-known to ordinary technicians in the field. According to the following examples, it is not difficult to successfully implement the present invention with minor modifications and variations according to specific circumstances, such as changing the culture temperature and the infection amount, etc. These modifications and variations are all within the scope of the claims of the present invention.

[0045] Example 1: In vitro culture of Myxosoma portunus

[0046] (1) Collect fresh diseased crabs, rinse the surface of the crabs with pure water, and then place them in 75% alcohol for 30 min to kill surface pathogens. Transfer them to a laminar flow hood, carefully cut open the abdomen and claws of the diseased crabs with sterilized surgical scissors, collect the muscle of the diseased crabs into a 2 mL Eppendorf tube, add 1:1 cell cryopreservation solution, mix well, and store in a -80 °C refrigerator to prevent the inability to collect Myxosoma portunus spores due to the decay of the diseased crabs.

[0047] (2) Prepare cell culture medium: DMEM medium (purchased from Zhongke Maichen) containing 10% FBS (purchased from Lonsera) and 1% double antibody (purchased from Xinsaimi). Take out RK-13 cells (purchased from ATCC, USA) from liquid nitrogen and quickly thaw them; after centrifugation at 1500 g for 5 min, add 4 mL of cell culture medium and transfer it to a T25 cell culture flask, and culture it under the conditions of 37 °C and 5% CO2. After growing into a confluent monolayer, digest it with 0.25% trypsin (purchased from Xinsaimi) and perform cell passage. After obtaining a sufficient number of RK-13 cells, digest them with 0.25% trypsin, resuspend the cells with the above medium, and transfer them to a 24-well plate to continue culturing until the cells grow into a monolayer.

[0048] (3) Add the collected diseased crab tissues to 30 times the volume of PBS for homogenization, and break them with a 27G needle, repeating 2 times. After breaking, filter the tissue fragments through a 5 μm sterile filter membrane. Centrifuge the filtrate at 5000 g for 5 min, resuspend the spores with PBS, and store them at 4 °C for later use.

[0049] (4) Adjust the spore concentration to 1×10 8 spores per milliliter of medium with cell culture medium.

[0050] (5) Add the spores in step (4) to the monolayer cells plated in the 24-well plate in step (2), adding 1 mL to each well. Continuously culture in an incubator at 37 °C and 5% CO2.

[0051] (6) Change the medium once every 2 days, aspirate 2 / 3 of the old medium, and supplement an equal amount of fresh medium.

[0052] Starting from the first day after infection, infected cells were collected every two days, and the newly produced spores at the sporulation stage and mature spores in the cells were counted. The specific method is as follows: Each time samples were collected, the cell culture medium in 3 wells of a 24-well plate was removed, 0.5 mL of PBS was added to each well, and this was repeated 2 times to remove exogenous spores on the cell surface. 20 μL of 0.25% trypsin was added to each well. After the cells were digested, they were transferred to a 1.5 mL EP tube, centrifuged at 10,000 g for 10 min, 500 μL of RIPA lysis buffer was added, and the cells were pipetted for 2 min to lyse the cells and release the spores inside the cells. After dilution with PBS buffer containing 10 μg / mL Direct Yellow 96, the number of mature spores was counted using a cell counting chamber under a fluorescence microscope, and the total number of spores was counted using a bright-field microscope. The results are as Figure 1 shown, indicating that Myxidium eriocheiris can infect RK-13 cells and can proliferate to produce a large number of spores.

[0053] After culturing for 13 days or more, the spores inside the cells can mature in large numbers. The cell infection rate of this method is above 90%. The mature spores inside the cells were stained with Direct Yellow 96 dye, and the mature spores were observed to be labeled green under a fluorescence microscope, as Figure 3 shown.

[0054] Example 2: In vitro continuous culture of Myxidium eriocheiris

[0055] (1) Fresh diseased crabs were collected, the surface of the crab body was rinsed with pure water, and then placed in 75% alcohol for 30 min to kill surface pathogens. Transferred to a laminar flow hood, the abdomen and crab claws of the diseased crab were carefully cut open with sterilized surgical scissors, the muscles of the diseased crab were collected into a 2 mL EP tube, mixed with 1:1 cell cryopreservation solution, and stored in a -80 °C refrigerator to prevent the inability to collect Myxidium eriocheiris spores due to the decay of the diseased crab.

[0056] (2) Prepare cell culture medium: DMEM medium (purchased from Zhongke Maichen) containing 10% FBS (purchased from Lonsera) and 1% double antibody (purchased from Xinsaimi). Take out RK-13 cells (purchased from ATCC, USA) from liquid nitrogen and quickly thaw them; after centrifuging at 1500 g for 5 min, add 4 mL of cell culture medium and transfer it to a T25 cell culture flask, and culture it under the conditions of 37 °C and 5% CO2. After growing into a confluent monolayer, digest it with 0.25% trypsin (purchased from Xinsaimi) and perform cell passage. After obtaining a sufficient number of RK-13 cells, digest them with 0.25% trypsin, resuspend the cells with the above medium, and transfer them to a 24-well plate to continue culturing until the cells grow into a monolayer.

[0057] (3) Add the collected diseased crab tissues to PBS at 30 times the volume for homogenization, and use a 27G needle to break them, repeating 2 times. After breaking, filter the tissue fragments through a 5μm sterile filter membrane. Centrifuge the filtrate at 5000g for 5 min, resuspend the spores with PBS, and store at 4°C for later use.

[0058] (4) Adjust the spore concentration to 1×10 8 spores per milliliter of the cell culture medium.

[0059] (5) Add the spores from step (4) to the monolayer cells plated in the 24-well plate in step (2), adding 1 mL to each well. Continuously culture in an incubator at 37°C and 5% CO2.

[0060] (6) Change the medium once every 2 days. Aspirate 2 / 3 of the old medium and supplement an equal amount of fresh medium.

[0061] (7) Subculture: Approximately 13 days after infection in step (6), remove the medium on the cell surface and slowly add 0.5 mL of PBS buffer to each well, repeating 2 times, to remove the exogenous spores purified from the diseased crab muscle and ensure that the obtained spores are those proliferated within RK-13 cells.

[0062] (8) Use trypsin to digest the adherent cells in the 24-well plate in step (7), resuspend the cells with PBS, centrifuge at 10000g for 10 min, add 5 mL of RIPA lysis buffer (purchased from Solarbio) to lyse the cell pellet, and continuously pipette for 10 min. After lysis, add 30 ml of PBS for dilution and continuously pipette for 5 min, then centrifuge at 5000g for 15 min (the supernatant contains immature spores). Collect the mature spore pellet, wash the surface lysis buffer with PBS 2 times, and centrifuge at 5000g for 10 min. This step can completely release the spores inside the cells and remove cell debris and immature spores.

[0063] (9) Resuspend the spore pellet with cell culture medium and adjust to 7×10 5 spores of H. eriocheiris per milliliter, and reinfect the RK-13 cells plated in the 24-well plate, adding 1 milliliter to each well.

[0064] The counting results of the mature spores are as Figure 2 shown (counted according to the method of Example 1), Figure 2 indicating that H. eriocheiris can be subcultured.

[0065] Example 3: In vitro continuous culture of H. eriocheiris

[0066] (1) Collect fresh diseased crabs, rinse the surface of the crabs with pure water, and then place them in 75% alcohol for 30 minutes to kill surface pathogens. Transfer them to a laminar flow hood, carefully cut open the abdomen and pincers of the diseased crabs with sterilized surgical scissors, collect the muscle of the diseased crabs into a 2 mL Eppendorf tube, add 1:1 cell cryopreservation solution, mix well, and store at -80 °C in the refrigerator to prevent the inability to collect spores of Myxosoma portunus due to the decay of the diseased crabs.

[0067] (2) Prepare cell culture medium: DMEM medium (purchased from Zhongke Maichen) containing 10% FBS (purchased from Lonsera) and 1% double antibody (purchased from Xinsaimi). Take out RK-13 cells (purchased from ATCC, USA) from liquid nitrogen and quickly thaw them; after centrifugation at 1500 g for 5 minutes, add 4 mL of cell culture medium and transfer it to a T25 cell culture flask, and culture it under the conditions of 37 °C and 5% CO2. After growing into a confluent monolayer, digest it with 0.25% trypsin (purchased from Xinsaimi) and perform cell passage. After obtaining a sufficient number of RK-13 cells, digest them with 0.25% trypsin, resuspend the cells with the above medium, and transfer them to a 24-well plate to continue culturing until the cells grow into a monolayer.

[0068] (3) Add the collected diseased crab tissues to 30 times the volume of PBS and homogenize them, and use a 27G needle to break them, repeating 2 times. After breaking, filter the tissue fragments through a 5 μm sterile filter membrane. Centrifuge the filtrate at 5000 g for 5 minutes, resuspend the spores with PBS, and store at 4 °C for later use.

[0069] (4) Adjust the spore concentration to 1×10 8 spores per milliliter of culture medium with cell culture medium.

[0070] (5) Add the spores in step (4) to the monolayer cells plated in the 24-well plate in step (2), adding 1 mL to each well. Continuously culture in an incubator at 37 °C and 5% CO2.

[0071] (6) Change the medium once every 2 days, aspirate 2 / 3 of the old medium, and supplement an equal amount of fresh medium.

[0072] (7) Subculture: Aspirate the old cell culture medium infected for more than 6 days in step (6), add 0.5 mL of PBS buffer to each well, wash the medium on the cell surface, repeating 2 times. Add 20 μL of trypsin to each well, and after the cells are digested, collect all the cells and mix them with uninfected RK-13 cells at a ratio of 1:1, and then replate them in a 24-well plate and continue culturing in an incubator at 37 °C and 5% CO2.

[0073] The bright field photograph of the proliferation of Myxosoma portunus in RK-13 cells at the 8th passage is as Figure 4 shown, and it can be clearly observed that the offspring produced by the proliferation of Myxosoma portunus in the cells.

[0074] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of RK-13 cells in in vitro culture of Spraguea carcinicola 2. The application according to claim 1, characterized in that RK-13 cells are used as infected cells for in vitro culture of Spraguea carcinicola.

3. A method for culturing Hematodinium sp. of swimming crab in vitro, characterized in that RK-13 cells are cultured to monolayer cells, and Spraguea carcinicola spores are added to the monolayer cells for in vitro culture of Spraguea carcinicola; during the in vitro culture of Spraguea carcinicola, liquid change is performed at regular intervals, and a part of the old culture medium is discarded each time, and an equal amount of fresh culture medium is supplemented.

4. The method for culturing Hematodinium perezi in vitro according to claim 3, characterized in that, The culture medium for culturing RK-13 cells to monolayer cells is DMEM medium containing fetal bovine serum and double antibiotics.

5. The method for in vitro culturing Hematodinium sp. of swimming crab according to claim 4, characterized in that, The content of fetal bovine serum is 9-11%, and the content of double antibiotics is 0.9-1.1%.

6. The method for in vitro culturing Hematodinium sp. of swimming crab according to claim 3, characterized in that, The in vitro culture of Spraguea carcinicola is carried out in a CO2 incubator at 36-38 °C and 4-6%.

7. The method for culturing Hematodinium perezi in vitro according to claim 3, characterized in that, The interval time for liquid change is 2-3 days; Or, when changing the liquid, 60-70% of the old culture medium is discarded.

8. The method for culturing Hematodinium perezi in vitro according to claim 3, characterized in that, The preparation method of Spraguea carcinicola spores is as follows: kill the pathogens on the surface of diseased crabs, then collect the muscles of diseased crabs, break the collected muscles of diseased crabs to obtain a tissue suspension, then filter and centrifuge the tissue suspension, and resuspend the spore precipitate with PBS to obtain.

9. The method for culturing Hematodinium perezi in vitro according to claim 3, characterized in that, It includes subculture of Spraguea carcinicola.

10. The method for culturing Hematodinium sp. of swimming crab in vitro according to claim 9, characterized in that, The subculture is as follows: Spraguea carcinicola is cultured in vitro for more than 12 days, exogenous spores are removed, the cells are lysed to release the Spraguea carcinicola spores in the cells, and the released Spraguea carcinicola spores are used to infect new RK-13 cells; Or, Spraguea carcinicola is cultured in vitro for more than 6 days, the cells are digested, and new RK-13 cells are added for in vitro culture.

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