Culture method and application of theileria annulata fission transformed cells
By gradually reducing the serum concentration and adjusting the medium ratio in the annular theileriosis cell culture, the cells were successfully adapted to low-serum culture, which solved the problems of high serum usage and high contamination risk in traditional methods, and achieved production cost reduction and scale. feasibility of chemical production.
Patent Information
- Application Number
- CN202411607000.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-12
- Publication Date
- 2025-01-14
AI Technical Summary
In the traditional culture method of circular Taylor worm schizos transformed cells, the use of high content of fetal calf serum leads to high production costs, unstable quality and high risk of microbial contamination.
By gradually reducing the concentration of newborn calf serum in a specific medium, from 10% to 3%, and adjusting the ratio of GB and RPMI1640 medium, theileria annular schizont-transformed cells were successfully adapted to low-serum culture.
It achieves the stability of cell growth and vitality under low serum conditions, reduces serum usage and production costs, and reduces the risk of microbial contamination. It is suitable for large-scale production of annular theileriosis vaccines.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cell biology, and in particular to a culture method and application of transformed cells of Theileria annularis schizonts. Background Art
[0002] Theileriasis annularis, also known as tropical theileriasis, is a tick-borne blood protozoan disease caused by the parasite Theileria annularis in bovine monocytes, lymphocytes and red blood cells. The disease is widespread in agricultural and pastoral areas. The typical clinical symptoms of animals infected are fever, swollen lymph nodes, jaundice, acute anemia, hemoglobinuria, etc. In severe cases, it can lead to animal death, causing huge economic losses to the animal husbandry industry.
[0003] Traditional culture of Theileria annularis schizont-transformed cells usually uses a culture medium containing 10%-20% fetal bovine serum, which provides cells with adhesion factors, growth factors, hormones and nutrients to ensure cell subculture. However, serum as an additive has the following disadvantages: 1) The serum composition is complex and uncertain, and there are differences in quality between batches, which increases the difficulty of vaccine production and quality control; 2) Serum is prone to cause contamination risks of microorganisms such as bacteria, fungi, exogenous viruses and mycoplasma, and the residual serum proteins in the vaccine are prone to cause allergic reactions in vaccinated animals, which increases the safety issues of vaccine production to a certain extent; 3) Serum itself is relatively expensive and has limited supply, which increases the difficulty of downstream purification of biological products while increasing production costs. Therefore, if the serum content can be reduced in the application of Theileria annularis vaccine, it is expected to overcome these process defects. Summary of the invention
[0004] One of the purposes of the present invention is to provide a method for culturing transformed cells of Theileria annularis schizonts with low serum, thereby providing a new and effective method for large-scale production of Theileria annularis disease vaccine.
[0005] The present invention provides a method for culturing transformed cells of Theileria annularis schizonts, which comprises: (1) taking the bovine lymphocytes transformed by the suspension culture of Theileria annularis schizonts and resuscitating them, and then placing them in a first culture medium containing 10% newborn calf serum for subculture and amplification culture; The first culture medium includes GB culture medium and RPMI1640 culture medium, with a pH value of 7.2-7.4, wherein the volume ratio of the GB culture medium to the RPMI1640 culture medium is 3:1; (2) Adapting cells to low serum culture, including the following steps: a) inoculating the schizont-transformed bovine lymphocytes amplified and cultured in step (1) into a second culture medium containing 8% newborn calf serum for subculture; the second culture medium comprises GB culture medium and RPMI1640 culture medium, with a pH value of 7.2-7.4, wherein the volume ratio of the GB culture medium to the RPMI1640 culture medium is 1:1; b) inoculating the cells obtained by subculture in step a) into a second culture medium containing 5% newborn calf serum for subculture; c) inoculating the cells obtained by subculture in step b) into a second culture medium containing 3% newborn calf serum for subculture; the GB culture medium is a solution of Gibco's A1627703 culture medium powder, and the concentration of the A1627703 culture medium powder in the GB culture medium is 17.5-18 g / L; the RPMI1640 culture medium is a solution of RPMI1640 culture medium powder, and the concentration of the RPMI1640 culture medium powder in the RPMI1640 culture medium is 24.5-25.5 g / L.
[0006] The present invention provides a method for culturing Theileria annularis schizont transformed cells in low serum. The method successfully domesticates the Theileria annularis schizont transformed cells to adapt to low serum culture by using a specific culture medium in combination with continuous adjustment of serum concentration, thereby greatly reducing the amount of bovine serum used and reducing production costs.
[0007] In the culture method of the present invention, the conditions for the subculture and amplification culture in step (1) are 37° C., 5% CO 2 , the number of subcultures is 2-3 times, and the culture time for each generation is 72 hours.
[0008] In step a) of the culture method of the present invention, the schizont-transformed bovine lymphocytes are in a logarithmic growth phase and the cell activity is greater than 90%.
[0009] The conditions for subculture in step (2) of the culture method of the present invention are: the inoculation density is 5×10 5 cells / mL, 80rpm, 37°C, 5% CO2 shaking culture, each generation culture time is 72h.
[0010] The present invention combines cell suspension culture technology, can effectively improve production efficiency, and can quickly and stably expand the production scale, providing basic materials for the research and development of attenuated vaccines for annular theileriasis.
[0011] The number of passages in steps a) and b) of the culture method of the present invention is 3 times.
[0012] The number of passages in step c) of the culture method of the present invention is ≥50.
[0013] The method of resuscitation in step (1) of the culture method of the present invention is: adding the thawed cells to the first culture medium, mixing them well, centrifuging them at 800 rpm for 5 minutes, and discarding the supernatant.
[0014] The present invention also provides application of the above-mentioned culture method in reducing the culture cost of Theileria annularis schizont transformed cells.
[0015] The present invention also provides application of the above-mentioned culture method in reducing the risk of microbial contamination during the culture of transformed cells of Theileria annularis schizonts.
[0016] The culture method of the present invention reduces the amount of serum used, thereby reducing production costs and reducing the risk of microbial contamination.
[0017] The present invention also provides application of the above-mentioned culture method in preparing annular theileriasis vaccine.
[0018] The method for preparing the vaccine for Theileria annularis disease can be found in: Studies on the cell culture and immunogenicity of Theileria annularis schizonts, Scientific Experiments, 1976, DOI: 10.16656 / j.issn.1673-4696.1976.02.003.
[0019] The beneficial effects of the present invention are at least: 1. The low-serum culture method of Theileria annularis schizont transformed cells of the present invention can effectively reduce the amount of bovine serum used in the culture medium, and the serum content only needs to be 3%; 2. The transformed cells of Theileria annularis schizonts cultured using low serum provided by the present invention can be continuously passaged for at least 50 generations in the low serum culture system; 3. The low-serum Theileria annularis schizont transformed cells harvested from domestication were stained with Giemsa, and the cell infection rate was close to 100%; the method for culturing Theileria annularis schizont transformed cells with low serum provided by the present invention not only improves the quality and yield of cells, but also reduces the cost of cell culture, and is more suitable for the large-scale production of Theileria annularis disease vaccines, providing strong technical support for the immune prevention of the disease. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is a photograph of the growth results of cells transformed with circular Taylor's schizonts, with 8% serum added and cultured for 72 hours.
[0021] Figure 2 This is a photograph of the growth results of cells transformed with circular Taylor's schizonts, which were cultured for 72 hours with 5% serum added.
[0022] Figure 3 This is a photograph of the growth results of cells transformed with circular Taylor's schizonts, which were cultured for 72 hours with 3% serum added.
[0023] Figure 4 This is a photograph of the growth results of the transformed cells of the ring-shaped Taylor schizont in the control group after 72 hours of culture.
[0024] Figure 5 This is a picture of the parasite infection in cells transformed by Theileria annularis schizonts under low serum culture conditions. DETAILED DESCRIPTION
[0025] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.
[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.
[0027] Example 1 This embodiment provides a method for culturing transformed cells of Theileria annularis schizonts, which is as follows: 1. Basic Methods 1. Materials and Methods 1.1 Cell source: The cells used were lymphocytes transformed from schizonts of Theileria annularis strain Kashi cultured in suspension in the Laboratory of Ectoparasites and Insect-borne Diseases of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, named TaKashi (recorded in: Establishment of a lymphocyte cell line infected with the Kashi strain of Theileria annularis and identification of its biological characteristics; Chinese Journal of Veterinary Science, 2019, 49(02): 144-151; DOI: 10.16656 / j.issn.1673-4696.2019.0021; Chinese Library Classification Number: S852.723, Document Code: A, Article Number: 1673-4696(2019)02-0144-08).
[0028] 1.2 Reagents: Newborn calf serum was purchased from a qualified supplier, RPMI 1640 and GB culture medium (dry powder) were purchased from Gibco, the item number of RPMI1640 is 31800022, and the item number of GB is A1627703; Giemsa stain was purchased from Beijing Solebow Company, and methanol was purchased from a domestic reagent; deionized water was produced by the laboratory itself.
[0029] Preparation of RPMI 1640 and GB culture medium: The culture medium needs to be prepared into liquid according to the instructions before use. The specific method is as follows: a. Place 25 g RPMI1640 dry powder culture medium and 17.7 g GB dry powder culture medium in containers respectively; b. Add 850mL of water for injection into each container, with the water temperature at about 30℃, and stir for 30 minutes; c. Add 2g and 2.32g of sodium bicarbonate to the solution containing RPMI1640 and GB, respectively. After they are completely dissolved, add water for injection to make the total solution volume 1L. d. Filter and sterilize with a 0.22 μm filter membrane, and store the solution in a sealed container at 2-8°C away from light; e. During use, if the pH value is not between 7.2 and 7.4, adjust the pH value to 7.2-7.4 with 0.5N sodium hydroxide or 0.5N hydrochloric acid.
[0030] 1.3 Instruments and equipment: biological clean bench (Sujing Antai Air Technology Co., Ltd.); pipette, high-speed centrifuge (Eppendorf); CO2 constant temperature incubator (Thermo Fisher Scientific); CO2 shaker incubator (Climo-Shaker ISF1-XC); cell analyzer (INNOVATIS); optical microscope (OLYMPUSDP71); cell smear centrifuge (Xiangyi TD3).
[0031] 1.4 Methods 1.4.1 Recovery of transformed cells from Theileria annularis schizonts Prepare a water bath and adjust the temperature to 37°C, and preheat the cell culture medium and serum at the same time; take out the frozen Theileria annularis schizont transformed suspension cells (TaKashi) in the cell cryopreservation tube from liquid nitrogen and quickly place them in a 37°C water bath, while continuously shaking rapidly to thaw the cells quickly and completely; under sterile conditions, add 5mL of culture medium (GB and RPMI1640 culture medium ratio is 3:1) to a 15mL centrifuge tube, then add the thawed cells thereto, gently mix, centrifuge at 800rpm for 5min at room temperature, and discard the supernatant; during the centrifugation process, prepare a cell culture medium containing 10% FBS and GB and RPMI1640 culture medium in a ratio of 3:1; take 6mL of cell culture medium to resuspend the cell pellet, inoculate the cells into a 25T cell bottle, place it in a 37°C, 5% CO2 constant temperature incubator for culture, and continuously passage the cells for 2-3 generations. After the cells form a dense monolayer, subsequent acclimation suspension culture is carried out.
[0032] 1.4.2 Adapting cells to low serum culture a) The cells in the logarithmic growth phase with cell activity > 90% of the schizont transformed cells were inoculated into a culture medium containing 8% and 5% newborn calf serum in a ratio of 1:1 GB and RPMI1640 and cultured for three generations. The specific inoculation density was 5×105 cells / mL, 80rpm, 37℃, 5% CO2 shaker culture, cells were cultured for 72h and then 5×10 5 The cell density of cells / mL was repeated for 3 generations, and the cell growth density and viability were recorded.
[0033] b) The cells obtained in step a) were inoculated in a culture medium containing 3% bovine serum and GB and RPMI1640 in a ratio of 1:1 for subculture at a seeding density of 5×10 5 cells / mL, 80rpm, 37°C, 5% CO2 shaking culture, each generation culture time is 72h, and the annular Theileria schizonts that are adapted to low serum culture are transformed into bovine lymphocytes.
[0034] 1.4.3 Verification of infection status of different generations of Theileria annularis schizont transformed cells cultured in low serum (3%) Collect cells of different generations that have adapted to low serum culture for 72 hours and have good growth status in 1.5mL EP tubes, place the slide in the cell smear centrifuge in advance, draw 200L of cell suspension into the centrifuge hole, centrifuge at 500rpm for 5min, at this time, the cells are evenly smeared on the slide, gently remove the slide from the centrifuge slot, and quickly spin dry the cells on the slide. Then fix with methanol for 5min, Giemsa stain for 30min, and observe the infection status of the intracellular parasites under an optical microscope.
[0035] 2. Results 2.1 At 8% and 5% serum content, the cell density can reach 4×10 6 cells / mL, cell viability can be maintained above 90%. The serum addition ratio is 8%, and the growth results of 72h culture are shown in the photo. Figure 1 The serum addition ratio is 5%, and the growth results after 72 hours of culture are shown in the photo. Figure 2 As shown in the figure, the cells have good morphology, neat edges, and are plump and translucent.
[0036] 2.2 The cells can grow normally in the presence of 3% newborn calf serum and maintain the original cell density and viability. The serum addition ratio is 3%. The growth results after 72 hours of culture are shown in the following figure. Figure 3 As shown, under electron microscope, the cells were round, translucent and evenly dispersed, which was different from the control group (cells were grown in cell culture medium containing GB and RPMI1640 medium with a ratio of 3:1 after recovery, Figure 4 ). Through the observation of cell culture for no less than 50 generations, it was found that the cells could grow stably at a concentration of 3% newborn calf serum, and the cell density and viability did not decrease significantly. Finally, the method of the present invention cultured a cell strain transformed from the schizont of Theileria annularis that could adapt to low serum culture.
[0037] 2.3 Giemsa staining was used to verify the intracellular parasite infection during the low serum acclimation process. The infection results of the 45th generation low serum cultured cells are shown in Figure 2. Figure 5 As shown, the infection of the parasites is close to 100%, indicating that the transformed cells of Theileria annularis schizonts can adapt to low serum culture while ensuring the infection of the parasites in the cells.
[0038] The present invention uses low-serum culture to domesticate theileria annularis schizonts to transform bovine lymphocytes, systematically optimizes the ratio of serum concentration to culture medium, and identifies the infection of parasites in schizont-transformed cells cultured in low serum. The experimental results show that cells can grow well in an optimized culture medium containing 3% newborn calf serum in a 1:1 ratio of GB and RPMI1640, and the cell density can reach 3.9×10 6 cells / mL, the activity is maintained above 90%, the 45th generation of low-serum cultured cells are subjected to Giemsa staining analysis, the parasite infection rate in the cells is close to 100%, and the cells are subcultured for at least 50 generations. The present invention reduces the cost of cell culture to a great extent by reducing the serum concentration to perform low-serum acclimation culture on the cells, promotes the large-scale production of the vaccine for annular theileriasis, and provides strong technical support for the immune prevention of the disease.
[0039] Comparative experiment This comparative experiment compares the effects of different culture media on the culture of Theileria annularis schizont transformed cells under low serum. The specific materials are the same as those in Example 1, and the specific method includes: 1. Recovery of transformed cells of Theileria annularis schizonts: the method is the same as that in Example 1.
[0040] 2. Culture under low serum: The cells in the logarithmic growth phase with cell activity > 90% of the schizont transformed cells were inoculated into different culture media containing 8% newborn calf serum for three passages, and the density and activity of cell growth were recorded. The specific inoculation and passage conditions were the same as those in step 1.4.2 of Example 1. The results are shown in Table 1.
[0041] Table 1 According to the results, adjusting the ratio of GB and RPMI1640 (3:1, 1:1, 1:3) will have unexpected effects on cell density and viability. The cells grow best in a culture medium with a GB and RPMI1640 ratio of 1:1, which can meet the requirements of cell production.
[0042] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.
Claims
1. A method for culturing transformed cells of Theileria annularis schizonts, characterized in that: include: (1) taking the bovine lymphocytes transformed by the suspension culture of Theileria annularis schizonts and resuscitating them, and then placing them in a first culture medium containing 10% newborn calf serum for subculture and amplification culture; The first culture medium includes GB culture medium and RPMI1640 culture medium, with a pH value of 7.2-7.4, wherein the volume ratio of the GB culture medium to the RPMI1640 culture medium is 3:1; (2) Adapting cells to low serum culture, including the following steps: a) inoculating the schizont-transformed bovine lymphocytes amplified and cultured in step (1) into a second culture medium containing 8% newborn calf serum for subculture; the second culture medium comprises GB culture medium and RPMI1640 culture medium, with a pH value of 7.2-7.4, wherein the volume ratio of the GB culture medium to the RPMI1640 culture medium is 1:1; b) inoculating the cells obtained by subculturing in step a) into a second culture medium containing 5% newborn calf serum for subculturing; c) The cells obtained by subculture in step b) are inoculated into a second culture medium containing 3% newborn calf serum for subculture; the GB culture medium is a solution of Gibco's A1627703 culture medium powder, and the concentration of the A1627703 culture medium powder in the GB culture medium is 17.5-18 g / L; the RPMI1640 culture medium is a solution of RPMI1640 culture medium powder, and the concentration of the RPMI1640 culture medium powder in the RPMI1640 culture medium is 24.5-25.5 g / L.
2. The culture method according to claim 1, characterized in that The conditions for the subculture and amplification in step (1) are 37° C., 5% CO 2 , the number of subcultures is 2-3 times, and the culture time for each generation is 72 hours.
3. The culture method according to claim 1 or 2, characterized in that In step a), the schizont-transformed bovine lymphocytes are in a logarithmic growth phase and the cell activity is greater than 90%.
4. The culture method according to any one of claims 1 to 3, characterized in that: The conditions for subculture in step (2) are: the inoculation density is 5×10 5 cells / mL, 80rpm, 37℃, 5% CO2 shaking culture, each generation culture time is 72h.
5. The culture method according to any one of claims 1 to 4, characterized in that: The number of passages in steps a) and b) was 3.
6. The culture method according to any one of claims 1 to 5, characterized in that: The number of passages in step c) is ≥ 50.
7. The culture method according to any one of claims 1 to 6, characterized in that: The method for resuscitation in step (1) is: adding the thawed cells to the first culture medium, mixing them well, centrifuging at 800 rpm for 5 minutes, and discarding the supernatant.
8. Use of the culture method according to any one of claims 1 to 7 in reducing the cost of culturing transformed cells of Theileria annularis schizonts.
9. Use of the culture method according to any one of claims 1 to 7 in reducing the risk of microbial contamination during the culture of transformed cells of Theileria annularis schizonts.
10. Use of the culture method according to any one of claims 1 to 7 in the preparation of a vaccine for theileriasis annularis.