Culture medium for high-density expression of FMDV3ABC antigen by escherichia coli and fermentation culture method
By optimizing E. coli culture medium and fermentation conditions, the problems of long preparation cycle and high cost of FMDV 3ABC antigens were solved, and high yield and high purity antigen expression was achieved, which promoted its large-scale production.
Patent Information
- Application Number
- CN202510490497.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-18
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, the preparation of FMDV 3ABC antigen has problems such as long culture cycle, high cost, low expression, and difficulty in purification, which restricts its large-scale production.
A culture medium for expressing FMDV 3ABC antigen in high density in E. coli, including basal medium and feed medium, is provided. By optimizing Ca2+ and Mg2+ concentrations, adding stabilizer polydimethylsiloxane aqueous emulsion and rose grass oil, combined with specific fermentation and culture conditions, adjusting pH and dissolved oxygen, and promoting the high density expression of FMDV 3ABC antigen in high density in E. coli.
The fermentation and culture time is shortened, the yield and purity of antigen products are improved, and efficient fermentation and culture of E. coli expressing FMDV 3ABC antigen is achieved.
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Abstract
Description
Technical Field
[0001] This application relates to the technical field of biomedicine, and particularly relates to a culture medium and a fermentation culture method for high-density expression of FMDV 3ABC antigen in Escherichia coli. Background Art
[0002] FMDV 3ABC antigen is the non-structural protein 3ABC of foot-and-mouth disease virus (FMDV), which is commonly used to distinguish infected animals from vaccinated animals. It plays a role in virus replication but does not participate in the formation of virus particles; infected animals will produce anti-3ABC antibodies, while vaccinated animals usually will not. Therefore, FMDV 3ABC antigen can be used for serological detection of anti-3ABC antibodies to distinguish natural infection from vaccination.
[0003] Currently, the preparation of FMDV 3ABC antigen faces problems such as long culture period, high cost, low expression level, and difficult purification, which restricts the large-scale production of FMDV 3ABC antigen. Summary of the Invention
[0004] To solve the above technical problems, this application provides a culture medium and a fermentation culture method for high-density expression of FMDV 3ABC antigen in Escherichia coli.
[0005] In a first aspect, this application provides a culture medium for high-density expression of FMDV 3ABC antigen in Escherichia coli, including a basal medium and a feeding medium; The basal medium is composed of components with the following concentrations: tryptone 7 - 9 g / L, yeast extract 15 - 17 g / L, glycerol 22 - 26 g / L, stabilizer 0.28 - 0.38 g / L, sodium ampicillin 0.05 - 0.15 g / L, NaCl 9 - 11 g / L, K2HPO4 3 - 5 g / L, MgSO4 0.3 - 0.5 g / L, CaCO3 3.5 - 5.3 g / L; the solvent is water; The feeding medium is composed of components with the following concentrations: glucose 460 - 540 g / L, peptone 8 - 12 g / L, yeast extract 4 - 6 g / L, glycerol 8 - 12 g / L, ammonium sulfate 8 - 12 g / L, stabilizer 0.28 - 0.38 g / L, sodium ampicillin 0.05 - 0.15 g / L; the solvent is water; In the basal medium and the feeding medium, the stabilizer is composed of a mixture of 10% polydimethylsiloxane aqueous emulsion and palmarosa oil with a weight ratio of 9 - 13:1 - 3.
[0006] The basal medium provided by the present application contains various nutrients, including inorganic salts, trace elements, and various nutrients required for growth, providing sufficient nutrients for the growth of bacterial strains. During the experiment, the applicant found that in the existing medium, the concentrations of Ca 2+ , Mg 2+ are often ignored, added in a fixed amount, or the addition ratio is not suitable for the fermentation culture of Escherichia coli for high-density expression of FMDV 3ABC antigen, resulting in problems such as insufficient cell membrane permeability, high plasmid loss rate, and increased inclusion body formation; during high-density fermentation, ion imbalance easily causes metabolic stress, affecting the yield and quality of products such as plasmid DNA, recombinant protein, and organic acid. The basal medium provided by the present application induces membrane lipid reorganization through Ca 2+ , combines with Mg 2+ to maintain membrane integrity and promote the excretion of metabolites; uses Mg 2+ to activate key enzymes of glycolysis (such as phosphofructokinase), and at the same time inhibits the activity of host proteases through Ca 2+ to reduce the degradation of target proteins; at the same time, the concentrations of the two in the medium are properly controlled, further improving the effect of fermentation culture.
[0007] The fed-batch medium after induction provided by the present application can adjust the pH value during the fermentation process, making the whole fermentation process in a good environment. The medium is rich in carbon source substances, which can be decomposed and metabolized by microorganisms to provide energy and carbon skeletons for cell growth, reproduction, and metabolite synthesis; and the medium can adjust the osmotic pressure of the fermentation broth, maintain the normal morphology and physiological functions of microbial cells, help microorganisms cope with osmotic stress, and prevent cell death due to water loss; at the same time, it can improve the rheological properties of the fermentation broth, reduce the viscosity of the fermentation broth, and improve the mass transfer efficiency, which is beneficial to the transfer of oxygen, nutrients, etc. to microbial cells, and the diffusion of metabolites from the cells into the fermentation broth, thereby promoting the growth and metabolism of microorganisms.
[0008] In addition, stabilizers are added to the basal medium and the feeding medium in this application. 10% polydimethylsiloxane aqueous emulsion in the stabilizer is used as an antifoaming agent. By reducing the surface tension of the liquid and destroying the stability of the foam film, the foam generated during fermentation can be eliminated, avoiding the occupation of the effective fermentation space by the foam and reducing the volume waste of the tank; reducing the risk of foam overflow; and at the same time reducing the obstruction of oxygen transfer by the foam layer or the entrapment of the bacterial cells, which affects the growth and metabolism of Escherichia coli expressing FMDV 3ABC antigen. Furthermore, the process conditions can be stabilized and the fermentation efficiency can be improved. Palmarosa oil can regulate the permeability of the cell membrane of Escherichia coli, making the nutrients in the medium easier to enter the cells and also contributing to the excretion of intracellular metabolites; this regulatory effect is beneficial to increasing the growth rate of Escherichia coli and the expression level of FMDV 3ABC antigen. When palmarosa oil and polydimethylsiloxane aqueous emulsion are used in combination, their unique chemical components have a synergistic effect with the emulsion, jointly promoting the effect of high-density expression of FMDV 3ABC antigen by Escherichia coli.
[0009] Preferably, in the basal medium, the concentration ratio of MgSO4 to CaCO3 is 0.35 - 0.45 g / L : 4.0 - 4.8 g / L.
[0010] Preferably, in the basal medium, the concentration ratio of MgSO4 to CaCO3 is 0.38 - 0.42 g / L : 4.2 - 4.6 g / L.
[0011] Furthermore, in the basal medium, the concentration ratio of MgSO4 to CaCO3 is 0.4 g / L : 4.4 g / L.
[0012] Preferably, the feeding medium is composed of the following components at the following concentrations: glucose 480 - 520 g / L, peptone 9 - 11 g / L, yeast extract 4.5 - 5.5 g / L, glycerol 9 - 11 g / L, ammonium sulfate 9 - 11 g / L, stabilizer 0.30 - 0.36 g / L, sodium ampicillin 0.07 - 0.12 g / L; the solvent is water.
[0013] Preferably, in the basal medium and the feeding medium, the stabilizer is composed of a mixture of 10% polydimethylsiloxane aqueous emulsion and palmarosa oil with a weight ratio of 10 - 12 : 1.5 - 2.5.
[0014] In a specific embodiment, in the stabilizer, the weight ratio of 10% polydimethylsiloxane aqueous emulsion to palmarosa oil can be 10 : 1.5, 11 : 1.5, 12 : 1.5, 10 : 2, 11 : 2, 12 : 2, 10 : 2.5, 11 : 2.5, 12 : 2.5.
[0015] In a second aspect, the present application provides the use of the above-mentioned culture medium in a fermentation culture method for high-density expression of FMDV 3ABC antigen by Escherichia coli.
[0016] In a third aspect, the present application provides a fermentation culture method for high-density expression of FMDV 3ABC antigen by Escherichia coli, which uses the above-mentioned culture medium for fermentation culture; specifically, it includes the following steps: Obtaining the strain: The constructed recombinant Escherichia coli strain expressing FMDV 3ABC protein is placed in an LB solid medium containing 0.05 - 0.15 g / L sodium ampicillin for culture to produce single colonies; Primary seed: The single colonies are inoculated into an LB liquid medium containing 0.05 - 0.15 g / L sodium ampicillin and shaken at a temperature of 36.8 - 37.2 °C. When the OD600 reaches 0.15 - 0.2, the primary seed is obtained and placed at 2 - 8 °C for later use; Fermentation of the bacterial liquid: The primary seed is inoculated into the basal medium and stirred at a pH of 6.8 - 7.2, a temperature of 36.8 - 37.2 °C, and a dissolved oxygen ≥ 50%; Induction of the bacterial liquid: When the OD600 reaches 3 - 5, an IPTG solution with a concentration of 0.8 - 1.2 M is added to make the concentration of the IPTG solution in the culture medium 0.19 - 0.29 g / L, and induction is carried out at a pH of 6.8 - 7.2, a temperature of 18 - 25 °C, and a dissolved oxygen ≥ 50%. At the same time, the feeding medium is added at a feeding rate of 10 - 18 ml / L per hour. When the OD600 value no longer increases, the bacterial sludge is harvested and the fermentation is completed; Harvesting and storing the bacterial sludge: The collected bacterial liquid is centrifuged to discard the supernatant, and the precipitate is collected; then it is stored at -20 °C.
[0017] Preferably, in the fermentation of the bacterial liquid, the dissolved oxygen condition is 65 - 75%.
[0018] Preferably, in the induction of the bacterial liquid, the temperature is 22 - 25 °C and the dissolved oxygen condition is 50 - 60%.
[0019] Preferably, the feeding rate of the feeding medium is 12 - 16 ml / L per hour In summary, the technical solution of the present application has the following effects: The present application provides a basal medium and a feeding medium for high-density expression of FMDV 3ABC antigen by Escherichia coli. By using the above-mentioned culture media in combination with a specific fermentation culture method, the fermentation culture time can be effectively shortened, and the obtained antigen product has a high yield and high purity.
[0020] In this application, by screening the concentrations of each raw material component in the basal medium (especially MgSO4 and CaCO3), and at the same time selecting a stabilizer composed of a 10% polydimethylsiloxane aqueous emulsion and citronella oil in a specific weight ratio for the basal medium and the feeding medium, the fermentation culture effect of Escherichia coli for high-density expression of FMDV 3ABC antigen is further improved.
[0021] In the fermentation culture method of Escherichia coli for high-density expression of FMDV 3ABC antigen in this application, by selecting an appropriate induction timing and dissolved oxygen control, the fermentation culture effect of Escherichia coli for high-density expression of FMDV 3ABC antigen is further improved. Description of the Drawings
[0022] Figure 1 SDS-PAGE electrophoresis diagram of the purified bacterial sludge harvested in Example 1 (M: protein marker; 1: Fermented and purified FMDV 3ABC antigen protein). Detailed Description of the Invention
[0023] The following further describes this application in detail with reference to examples, comparative examples, and performance detection tests. These examples should not be construed as limiting the scope claimed in this application.
[0024] The 10% polydimethylsiloxane aqueous emulsion (product number A5757) used in this application was purchased from Sigma; citronella oil (CAS number: 8014-19-5, product number S68183); sunflower seed oil (CAS number: 8001-21-6, product number S24927) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Examples
[0025] Example 1 Example 1 provides a medium and a fermentation culture method for Escherichia coli to highly express FMDV 3ABC antigen.
[0026] The fermentation culture method of Escherichia coli for high-density expression of FMDV 3ABC antigen in Example 1 is specifically as follows.
[0027] The basal medium consists of the following components in concentrations: tryptone 8 g / L, yeast extract 16 g / L, glycerol 24 g / L, stabilizer 0.33 g / L (the stabilizer is composed of a 10% polydimethylsiloxane aqueous emulsion and citronella oil mixed in a weight ratio of 11:2), sodium ampicillin 0.1 g / L, NaCl 10 g / L, K2HPO4 4 g / L, MgSO4 0.4 g / L, CaCO3 4.4 g / L; the solvent is water.
[0028] The fed-batch medium consists of components at the following concentrations: glucose 500 g / L, peptone 10 g / L, yeast extract 5 g / L, glycerol 10 g / L, ammonium sulfate 10 g / L, stabilizer 0.33 g / L (the stabilizer is composed of a 10% polydimethylsiloxane aqueous emulsion and palmarosa oil mixed at a weight ratio of 11:2), sodium ampicillin 0.1 g / L; the solvent is water.
[0029] Strain: Insert the highly conserved 3ABC protein gene of foot-and-mouth disease virus into the plasmid vector pET-21a(+), transform Escherichia coli, and prepare recombinant Escherichia coli expressing the 3ABC protein of foot-and-mouth disease virus; place the constructed recombinant Escherichia coli strain expressing the FMDV 3ABC protein in an LB solid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 16 g / L) containing 0.1 g / L of the antibiotic sodium ampicillin for culture to produce single colonies.
[0030] Primary seed: Inoculate the single colony into an LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) containing 0.1 g / L of the antibiotic sodium ampicillin, and culture it with shaking at 37 °C. When the OD600 reaches 0.18, obtain the primary seed and place it at 2 - 8 °C for later use.
[0031] Fermentation of bacterial liquid: Inoculate the primary seed into the basal medium and culture it with stirring under the conditions of pH = 7, temperature of 37 °C, and dissolved oxygen of 65 - 75% until the OD600 reaches 4.
[0032] Induction of bacterial liquid: When the OD600 reaches 4, add IPTG at a concentration of 1 M to make the concentration of the IPTG solution in the medium 0.24 g / L (1 mM), and perform induction under the conditions of a temperature of 23 °C and a dissolved oxygen condition of 50 - 60%. At the same time, add the fed-batch medium at a feeding rate of 14 ml / L per hour. When the OD600 value no longer increases, harvest the bacterial sludge and complete the fermentation (in this example, the OD600 can reach 6 after 1 hour of induction, 10 after 2 hours, 25 after 6 hours of induction, and the OD600 value reaches 25.5 and no longer increases after 6.5 hours of induction).
[0033] Harvest and storage of bacterial sludge: Centrifuge the collected bacterial liquid at 4 °C and 6000 r / min for 30 min, discard the supernatant, and collect the precipitate; then store it at -20 °C.
[0034] Homogenize and crush the harvested bacterial sludge (i.e., FMDV 3ABC antigen) (using a homogenizer), centrifuge (centrifuge at 4 °C and 12000 r / min for 30 minutes), filter (0.8 μm - 0.45 μm), purify with a Ni column, and take a sample for SDS-PAGE electrophoresis. The electrophoresis pattern is asFigure 1 As shown, M represents protein marker; 1 represents the fermented and purified FMDV 3ABC antigen protein; the molecular weight of FMDV 3ABC antigen is 42 kDa); the purity of the target protein was calculated using gray scale analysis, and the purity was approximately 94.9%.
[0035] Examples 2 - 7 Examples 2 - 7 respectively provided a culture medium and a fermentation culture method for high - density expression of FMDV 3ABC antigen in Escherichia coli.
[0036] The differences between the above - mentioned examples and Example 1 are as follows: the concentrations of each component in the basal medium are different, as shown in Table 1 specifically.
[0037] Table 1 Concentrations of each component in the basal medium in Examples 1 - 7 In the above - mentioned examples, other process parameters are the same as those in Example 1.
[0038] Examples 8 - 11 Examples 8 - 11 respectively provided a culture medium and a fermentation culture method for high - density expression of FMDV 3ABC antigen in Escherichia coli.
[0039] The differences between the above - mentioned examples and Example 1 are as follows: the stabilizers in the basal medium and the feeding medium are different, as shown below.
[0040] In Example 8: the stabilizer is composed of a 10% polydimethylsiloxane aqueous emulsion and palmarosa oil mixed in a weight ratio of 9:3 (3:1).
[0041] In Example 9: the stabilizer is composed of a 10% polydimethylsiloxane aqueous emulsion and palmarosa oil mixed in a weight ratio of 13:1.
[0042] In Example 10: the stabilizer is composed of a 10% polydimethylsiloxane aqueous emulsion and palmarosa oil mixed in a weight ratio of 10:2.5.
[0043] In Example 11: the stabilizer is composed of a 10% polydimethylsiloxane aqueous emulsion and palmarosa oil mixed in a weight ratio of 12:1.5.
[0044] In the above - mentioned examples, other process parameters are the same as those in Example 1.
[0045] Examples 12 - 14 Examples 12 - 14 respectively provided a culture medium and a fermentation culture method for high - density expression of FMDV 3ABC antigen in Escherichia coli.
[0046] The differences between the above embodiments and Embodiment 1 are as follows: the conditions for bacterial liquid fermentation and induction are different, as specifically shown below.
[0047] In Embodiment 12: Bacterial liquid fermentation: Inoculate the primary seed into the basal medium, and perform stirring culture under the conditions of pH = 7, temperature of 37 °C, and dissolved oxygen of 50 - 60%.
[0048] In Embodiment 13: Bacterial liquid fermentation: Inoculate the primary seed into the basal medium, and perform stirring culture under the conditions of pH = 7, temperature of 37 °C, and dissolved oxygen of 80 - 90%.
[0049] In Embodiment 14: Bacterial liquid induction: When OD600 reaches 4, add IPTG with a concentration of 1 M to make the concentration of IPTG solution in the medium 0.24 g / L (1 mM), and perform induction under the conditions of temperature of 23 °C and dissolved oxygen condition of 65 - 75%. At the same time, add the feeding medium at a feeding rate of 14 ml / L per hour. When the OD600 value no longer increases, the fermentation is completed; centrifuge the harvested fermentation broth at 4 °C and 6000 r / min for 30 min, discard the supernatant, weigh the precipitate, and resuspend and wash it once with PBS (0.01 mol / L, pH value 7.4) at a mass ratio of 1:10; centrifuge the fully resuspended liquid at 4 °C and 6000 r / min for 30 minutes, discard the supernatant, and collect and weigh the precipitate, which is the harvested bacterial sludge.
[0050] In the above embodiments, other process parameters are the same as those in Embodiment 1.
[0051] Comparative Example Comparative Examples 1 - 5 Comparative Examples 1 - 5 respectively provide a medium and a fermentation and culture method for expressing FMDV 3ABC antigen by Escherichia coli.
[0052] The differences between the above comparative examples and Embodiment 1 are specifically shown as follows.
[0053] In Comparative Example 1: In the basal medium, the concentration of MgSO4 is 4.4 g / L; the concentration of CaCO3 is 0.4 g / L.
[0054] In Comparative Example 2: In the basal medium and the feeding medium, the stabilizer is 10% polydimethylsiloxane aqueous emulsion.
[0055] In Comparative Example 3: In the basal medium and the feeding medium, the stabilizer is composed of a mixture of 10% polydimethylsiloxane aqueous emulsion and sunflower oil with a weight ratio of 11:2.
[0056] In Comparative Example 4: In the basal medium and the feeding medium, the stabilizer is composed of a mixture of 10% polydimethylsiloxane aqueous emulsion and sunflower oil with a weight ratio of 2:11.
[0057] In Comparative Example 5: Bacterial liquid fermentation: The primary seed was inoculated into the basal medium and cultured with stirring under the conditions of pH = 7, temperature of 37°C, and dissolved oxygen of 35 - 45%. Bacterial liquid induction: When OD600 reached 4, IPTG with a concentration of 1 M was added to make the concentration of IPTG solution in the medium 0.24 g / L (1 mM), and induction was carried out at a temperature of 23°C and a dissolved oxygen condition of 35 - 45%. Meanwhile, the feeding medium was added at a feeding rate of 14 ml / L per hour. When the OD600 value no longer increased, the bacterial sludge was harvested and the fermentation was completed.
[0058] Other process parameters in the above comparative examples were the same as those in Example 1.
[0059] Performance detection test (1) Fermentation duration: According to the fermentation culture methods in the examples and comparative examples, the time when the OD600 values reached 6, 10, 25, and when the OD600 value no longer changed during the bacterial liquid induction process was detected and recorded (detected every 30 minutes).
[0060] (2) Bacterial sludge yield: Bacterial sludge yield = weight of harvested bacterial sludge precipitate / fermentation volume.
[0061] (3) Purity: The harvested bacterial sludge was homogenized and broken (homogenizer), centrifuged (centrifuged at 4°C and 12,000 r / min for 30 minutes), filtered (0.8 μm - 0.45 μm), and purified by Ni column. Samples were taken for SDS - PAGE electrophoresis, and the purity of the target protein was calculated using gray - scale analysis.
[0062] The detection results are shown in Table 2.
[0063] Table 2 Performance detection results of the fermentation culture methods in the examples and comparative examples Combined with the detection results in Table 2, it can be seen that in the fermentation culture method for high - density expression of FMDV 3ABC antigen using the culture medium provided by the present application, the fermentation culture duration can be shortened, and the obtained antigen product has a high yield and high purity.
[0064] In the basal medium of Comparative Example 1, the concentrations of MgSO4 and CaCO3 did not match, and the fermentation culture effect of Escherichia coli expressing FMDV 3ABC antigen was poor. In contrast, in Examples 1, 4-7 of the present application, by selecting specific concentrations of MgSO4 and CaCO3 and adding them to the basal medium, the fermentation culture time can be effectively shortened, and the yield and purity of the antigen product can be improved. At the same time, in Examples 1-3 of the present application, by controlling the amounts of the raw material components in the basal medium within a suitable range, the components cooperate synergistically, effectively ensuring the fermentation culture effect of high-density expression of FMDV 3ABC antigen by Escherichia coli.
[0065] In Comparative Example 2, the stabilizer in the basal medium and the feeding medium was 10% polydimethylsiloxane aqueous emulsion. In Comparative Example 3, the stabilizer in the basal medium and the feeding medium was composed of a mixture of 10% polydimethylsiloxane aqueous emulsion and sunflower oil with a weight ratio of 11:2. In Comparative Example 4, the stabilizer in the basal medium and the feeding medium was composed of a mixture of 10% polydimethylsiloxane aqueous emulsion and sunflower oil with a weight ratio of 2:11, and the fermentation culture effect of Escherichia coli expressing FMDV 3ABC antigen was poor. In contrast, in Examples 1, 8-11 of the present application, by selecting a stabilizer composed of a mixture of 10% polydimethylsiloxane aqueous emulsion and palmarosa oil with a weight ratio of 9-13:1-3, the fermentation culture time can be effectively shortened, and the yield and purity of the antigen product can be improved.
[0066] In Comparative Example 5, the dissolved oxygen condition during the fermentation and induction of the bacterial liquid was lower than 50%, and the fermentation culture effect of Escherichia coli expressing FMDV 3ABC antigen was poor. In contrast, in Examples 1, 12-14 of the present application, by controlling the dissolved oxygen condition during the fermentation and induction of the bacterial liquid above 50%, the fermentation culture effect of Escherichia coli expressing FMDV 3ABC antigen was greatly improved.
[0067] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.
Claims
1. A culture medium for high-density expression of FMDV 3ABC antigen in Escherichia coli, characterized in that, It includes a basal medium and a feeding medium; The basal medium is composed of components with the following concentrations: tryptone 7 - 9 g / L, yeast extract 15 - 17 g / L, glycerol 22 - 26 g / L, stabilizer 0.28 - 0.38 g / L, sodium ampicillin 0.05 - 0.15 g / L, NaCl 9 - 11 g / L, K2HPO4 3 - 5 g / L, MgSO4 0.3 - 0.5 g / L, CaCO3 3.5 - 5.3 g / L; the solvent is water; The feeding medium is composed of components with the following concentrations: glucose 460 - 540 g / L, peptone 8 - 12 g / L, yeast extract 4 - 6 g / L, glycerol 8 - 12 g / L, ammonium sulfate 8 - 12 g / L, stabilizer 0.28 - 0.38 g / L, sodium ampicillin 0.05 - 0.15 g / L; the solvent is water; In the basal medium and the feeding medium, the stabilizer is composed of a mixture of 10% polydimethylsiloxane aqueous emulsion and palmarosa oil with a weight ratio of 9 - 13:1 - 3.
2. The culture medium for high-density expression of FMDV 3ABC antigen according to claim 1, characterized in that, In the basal medium, the concentration ratio of MgSO4 to CaCO3 is 0.35 - 0.45 g / L:4.0 - 4.8 g / L.
3. The culture medium for high-density expression of FMDV 3ABC antigen in Escherichia coli according to claim 2, characterized in that, In the basal medium, the concentration ratio of MgSO4 to CaCO3 is 0.38 - 0.42 g / L:4.2 - 4.6 g / L.
4. The culture medium for high-density expression of FMDV 3ABC antigen in Escherichia coli according to claim 1, characterized in that, The feeding medium is composed of components with the following concentrations: glucose 480 - 520 g / L, peptone 9 - 11 g / L, yeast extract 4.5 - 5.5 g / L, glycerol 9 - 11 g / L, ammonium sulfate 9 - 11 g / L, stabilizer 0.30 - 0.36 g / L, sodium ampicillin 0.07 - 0.12 g / L; the solvent is water.
5. The culture medium for high-density expression of FMDV 3ABC antigen in Escherichia coli according to claim 1, characterized in that, In the basal medium and the feeding medium, the stabilizer is composed of a mixture of 10% polydimethylsiloxane aqueous emulsion and palmarosa oil with a weight ratio of 10 - 12: 1.5 - 2.
5.
6. Use of the medium according to any one of claims 1 - 5 in the fermentation culture method for high - density expression of FMDV 3ABC antigen by Escherichia coli.
7. A fermentation culture method for high-density expression of FMDV 3ABC antigen by Escherichia coli, characterized in that, Fermentation culture is carried out using the medium according to any one of claims 1 - 5; specifically, it includes the following steps: Strain acquisition: The constructed recombinant Escherichia coli strain expressing FMDV 3ABC protein is cultured in an LB solid medium containing 0.05 - 0.15 g / L of sodium ampicillin to produce single colonies; Primary seed: The single colonies are inoculated into an LB liquid medium containing 0.05 - 0.15 g / L of sodium ampicillin and shaken at a temperature of 36.8 - 37.2 °C. When the OD600 reaches 0.15 - 0.2, the primary seed is obtained and stored at 2 - 8 °C for later use; Fermentation of the bacterial liquid: The primary seed is inoculated into the basal medium and stirred and cultured under the conditions of pH = 6.8 - 7.2, temperature of 36.8 - 37.2 °C, and dissolved oxygen ≥ 50%; Bacterial liquid induction: When the OD600 reaches 3 - 5, add an IPTG solution with a concentration of 0.8 - 1.2 M so that the concentration of the IPTG solution in the culture medium is 0.19 - 0.29 g / L. Induce under the conditions of pH = 6.8 - 7.2, temperature = 18 - 25°C, and dissolved oxygen ≥ 50%. At the same time, add the feeding medium at a feeding rate of 10 - 18 ml / L per hour. When the OD600 value no longer increases, harvest the bacterial sludge and complete the fermentation; Harvest and storage of bacterial sludge: Centrifuge the collected bacterial liquid to discard the supernatant and collect the precipitate; then store it at -20°C.
8. The fermentation culture method for high-density expression of FMDV 3ABC antigen by Escherichia coli according to claim 7, characterized in that, During the fermentation of the bacterial liquid, the dissolved oxygen condition is 65 - 75%.
9. The fermentation and culture method for high-density expression of FMDV 3ABC antigen by Escherichia coli according to claim 7, characterized in that, During the induction of the bacterial liquid, the temperature is 22 - 25°C and the dissolved oxygen condition is 50 - 60%.
10. The fermentation culture method for high-density expression of FMDV 3ABC antigen by Escherichia coli according to claim 7, characterized in that, The feeding rate of the feeding medium is 12 - 16 ml / L per hour.