A method for increasing the proportion of supercoiled plasmids in production and uses thereof

Abstract

The application belongs to the technical field of veterinary preparation, and specifically discloses a production method for improving the proportion of supercoils in plasmids and application thereof. The application forms a combined culture medium system through a specifically constructed basic culture medium, a feed medium and an expression culture medium, and cooperates with a specifically constructed 5-stage culture mode, so that sufficient nutrient substances can be provided for rapid growth of the bacterial cells, the yield of plasmid DNA is improved, and the damage to the plasmid DNA is reduced, so that the proportion of supercoils in the finally obtained plasmid is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of veterinary preparation technology, specifically relating to a production method for increasing the proportion of supercoiled plasmids and its applications. Background Technology

[0002] Nucleic acid vaccines involve directly introducing a foreign gene encoding a specific antigen protein into animal cells after recombination with a plasmid. The host cell's transcription system then synthesizes the antigen protein, inducing an immune response in the host to prevent and treat diseases. Veterinary vaccine development has consistently focused on safety, efficiency, and cost-effectiveness. The classic first-generation inactivated / live vaccine production process is mature, and second-generation subunit genetically engineered vaccines are already available. DNA nucleic acid vaccines belong to the third generation of vaccine technology. Compared to the first two generations, DNA nucleic acid vaccines offer numerous advantages.

[0003] First, in terms of biosafety, DNA nucleic acid vaccines pose no threat of virulence reversion, significantly reducing the chance of bacterial and viral recombination in animals and preventing the release of toxins into the environment, thus facilitating disease eradication. Second, experiments have shown that DNA nucleic acid vaccines can generate cross-protection between different strains of the same species, and the process of producing combination vaccines is much simpler than that of traditional vaccines. Third, DNA nucleic acid vaccines have higher production efficiency, yielding tens of millions of doses per batch with minimal batch-to-batch variation, resulting in more stable and controllable product quality. Furthermore, the production process is relatively standardized, does not involve live viruses with high biosafety levels, and facilitates rapid scale-up production. Fourth, regarding transportation and storage, since the core of a DNA vaccine is plasmid DNA, which has good stability, cold chain transportation is unnecessary, and storage is convenient.

[0004] Although DNA nucleic acid vaccines have shown significant advantages in inducing broad-spectrum immune responses, R&D and production cycles, and product stability, their transformation from laboratory construction to large-scale production and application, especially in the field of animal disease prevention and control, still faces many key technological bottlenecks that restrict industrialization. Veterinary vaccines, due to the specific nature of their target populations and market demands, pose stringent challenges to production cost control and product quality standards. This requires that their large-scale production processes must reduce overall costs while fully ensuring the safety and efficacy of the vaccine. Achieving a synergistic balance between high unit volume yield and a high supercoil configuration ratio of plasmid DNA in large-scale fermentation production is currently recognized as one of the core technological challenges in the field.

[0005] While existing technologies can employ high-density fermentation strategies to obtain large amounts of bacterial biomass, increased cell density does not necessarily equate to a corresponding increase in plasmid yield. This is because, on the one hand, conventional culture media are poorly designed and lack specific nutrient supply to the plasmid biosynthetic pathway, easily leading to excessive accumulation of metabolic byproducts (such as acetic acid). This, in turn, inhibits the physiological activity of the host bacteria and the stable replication of the plasmid, resulting in significant loss of the target product. On the other hand, the supercoil conformation, a key quality attribute determining the in vivo efficacy of DNA vaccines, is highly susceptible to fluctuations in fermentation conditions.

[0006] In current processes, factors such as imprecise control of plasmid replication timing, stress-induced temperature variations during culture and induction, and inappropriate feeding strategies often lead to the irreversible conversion of supercoiled DNA into inactive open-circular or linear forms, severely weakening vaccine efficacy. Therefore, developing a novel culture medium and method suitable for the large-scale production of veterinary DNA nucleic acid vaccines is crucial to overcoming the current technical barriers to yield and quality in high-density fermentation. Simultaneously improving plasmid DNA yield and supercoil ratio is essential for reducing vaccine production costs and ensuring final product quality, representing significant industrial application value and an urgent technological innovation need. Summary of the Invention

[0007] To address the aforementioned shortcomings in the prior art, this invention provides a production method for increasing the supercoil ratio in plasmids and its applications.

[0008] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide a production method for increasing the proportion of supercoiled plasmids, which includes the following steps: (1) Inoculate the recombinant Escherichia coli strain containing the duck circovirus Cap gene plasmid into the basic culture medium at a ratio of 1-5%, and use it as the production strain after cultivation; the basic culture medium includes the following components at the following concentrations: glucose 3-10 g / L, glycerol 5-10 g / L, yeast extract 3-10 g / L, tryptone 5-20 g / L, sodium chloride 5-15 g / L, potassium dihydrogen phosphate 2-5 g / L, dipotassium hydrogen phosphate 10-20 g / L, magnesium sulfate 0.1-5 g / L, citric acid 0.2-2 g / L, defoamer 0.005-2%, trace element solution 0.001-0.01%, and antibiotic solution 0.005-0.05%; (2) Inoculate the production strain into the basic culture medium at a ratio of 1-5% and continue to culture at 32-35℃; (3) Step (2) Cultivate to OD 600 When the temperature reaches 20-30°C, supplemental feeding should be performed using fed culture medium at a rate of 0.5-1.5 ml / OD per liter of culture volume per hour.600 The culture was continued at 32-35℃. The fed culture medium consisted of the following components at the following concentrations: glucose 1-5 g / L, glycerol 100-1000 g / L, yeast extract 3-10 g / L, tryptone 5-20 g / L, sodium chloride 5-15 g / L, potassium dihydrogen phosphate 2-5 g / L, dipotassium hydrogen phosphate 10-20 g / L, magnesium sulfate 0.1-5 g / L, citric acid 0.2-2 g / L, defoamer 0.005-2%, trace element solution 0.001-0.01%, and antibiotic solution 0.005-0.05%. (4) Step (3) Cultivate to OD 600 When the temperature reaches 40-60°C, supplemental feeding should be performed using fed culture medium at a rate of 0.5-1.5 ml / OD per liter of culture volume per hour. 600 During the cultivation process, the temperature is increased to 38-40℃ at a rate of 1-2℃ / h; (5) After heating to the predetermined temperature in step (4), stop adding the feeding medium, and then add the expression medium at a rate of 1~2 mL / L·h, and continue culturing at 38~40℃; the expression medium includes the following components at the following concentrations: glycerol 100~1000 g / L, yeast extract 3~10 g / L, tryptone 5~20 g / L, sodium chloride 5~15 g / L, magnesium sulfate 0.1~5 g / L, citric acid 0.2~2 g / L, astragalus extract 0.1~10 g / L, codonopsis extract 0.01~10 g / L, and antibiotic solution 0.005~0.05%; (6) After the plasmid expression in step (5) is completed, the temperature is lowered to 15~18℃, and then the expression medium is added at a rate of 3~5mL / L·h to culture for 2~5h to obtain a plasmid with a high supercoil ratio.

[0009] Furthermore, in step (1), the culture temperature is 35~37℃ and the culture time is 10~16h.

[0010] Furthermore, in step (2), the inoculation ratio of the production strain is 1-2%, the culture temperature is 32-35℃, the pH is 7.0-7.5, and the dissolved oxygen rate is 30-35%.

[0011] Furthermore, the feeding rate in step (3) is 0.5~1.0 ml / OD. 600 The culture temperature is 32~35℃, the pH is 7.0~7.5, and the dissolved oxygen rate is 30~35%.

[0012] Furthermore, the feeding rate in step (4) is 0.5~1.0 ml / OD. 600 During the cultivation process, the temperature was increased to 40℃ at a rate of 2℃ / h, the pH of the cultivation system was 7.0~7.5, and the dissolved oxygen rate was 30~35%.

[0013] Furthermore, in step (5), the feeding rate is 2 mL / L·h, the culture temperature is 40℃, the pH is 7.0~7.5, and the dissolved oxygen rate is 30~35%.

[0014] Furthermore, the defoamer is defoamer 204.

[0015] Furthermore, the trace element solutions in the basal and supplemental culture media include the following components at the following concentrations: Cobalt chloride 0.1~0.4 g / L, calcium chloride 2~5 g / L, copper chloride 0.05~0.3 g / L, boric acid 0.2~0.5 g / L, manganese chloride 0.8~2.5 g / L, sodium molybdate 0.1~0.4 g / L, zinc acetate 0.5~2.0 g / L, ferric citrate 5~12 g / L.

[0016] Furthermore, the antibiotic solution in the basal culture medium, feed medium, and expression medium is an ampicillin solution with a concentration of 100-120 g / L.

[0017] Another object of the present invention is to provide a plasmid with a high hypercolic ratio, which is prepared by the above method.

[0018] Another object of the present invention is to provide the use of the above-described plasmid in the development of DNA nucleic acid vaccines.

[0019] The beneficial effects of this invention are: This invention utilizes basal culture medium, supplemented culture medium, and expression culture medium to meet the nutritional needs of *E. coli* at different stages and to achieve high-efficiency plasmid expression. The *Astragalus membranaceus* extract and *Codonopsis pilosula* extract added to the expression culture medium have a protective effect on the plasmid. The mechanism of action is as follows: *Astragalus membranaceus* polysaccharides and astragaloside in the *Astragalus membranaceus* extract can reduce plasmid DNA damage by inhibiting oxidative stress and regulating the expression of related genes, thereby maintaining the supercoiled structure of the plasmid DNA; while *Codonopsis pilosula* polysaccharides in the *Codonopsis pilosula* extract can reduce DNA damage caused by oxidative stress by scavenging free radicals such as reactive oxygen species, and synergistically work with *Astragalus membranaceus* polysaccharides to further stabilize the supercoiled structure of the plasmid DNA.

[0020] This invention employs a five-stage culture method in the high-density bacterial fermentation production of plasmid DNA, using different culture temperatures and media at different stages, instead of the conventional method of using the same temperature and conditions. In the first stage, the bacterial expansion culture is conducted at 32°C. Under these conditions, the bacteria can rapidly and stably accumulate biomass without prematurely initiating plasmid production, thus avoiding the metabolic burden caused by early plasmid replication. The second stage is the mid-exponential growth phase of the bacteria. Feeding during this stage aims to provide sufficient nutrients for rapid bacterial growth. In the third stage, the culture temperature is gradually increased to 40°C. This stage is the mid-to-late exponential growth phase of the bacteria, ensuring rapid bacterial growth while simultaneously triggering plasmid replication, obtaining a certain yield of plasmids without adversely affecting the bacteria. The fourth stage is the bacterial growth plateau phase and the stage of massive plasmid replication. During this stage, plasmid production reaches its peak, with a large amount of metabolites produced and accumulated. Using expression media reduces damage to the plasmid DNA, ensuring a high proportion of supercoiled plasmids in the final product. The fifth stage, lowering the temperature, can effectively slow down and stop the metabolic activities of the cells, prevent the plasmid from being degraded by nucleases, and at the same time maintain the integrity of the bacterial structure, so that the plasmid is retained in the bacteria to the greatest extent, which facilitates the collection of bacterial cells, subsequent purification operations, and the maintenance of the supercoiled structure of plasmid DNA. Attached Figure Description

[0021] Figure 1 Electrophoresis images of plasmid DNA prepared by different methods; where M: DNA maker; 1: Example 3; 2: Example 2; 3: Example 1; 4: Comparative Example 1; 5: Comparative Example 2; 6: Comparative Example 3; 7: Comparative Example 4; 8: Comparative Example 5. Detailed Implementation

[0022] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0023] The culture medium used in this invention and its preparation method are as follows: 1. Preparation of trace element solutions and antibiotic solutions Each liter of trace element solution comprises: 0.1–0.4 g cobalt chloride, 2–5 g calcium chloride, 0.05–0.3 g copper chloride, 0.2–0.5 g boric acid, 0.8–2.5 g manganese chloride, 0.1–0.4 g sodium molybdate, 0.5–2.0 g zinc acetate, and 5–12 g ferric citrate. Weigh each component, dissolve it thoroughly in pure water, accurately dilute to volume, filter using a 0.22 μm filter, and store at 2–8 °C for later use.

[0024] The antibiotic solution is 100 g / L ampicillin, fully dissolved in pure water, accurately diluted to volume, filtered using a 0.22 μm filter, and stored at 2-8℃ for later use.

[0025] 2. Preparation of basal culture medium Each liter of basal culture medium comprises: 3-10g glucose, 5-10g glycerol, 3-10g yeast extract, 5-20g tryptone, 5-15g sodium chloride, 2-5g potassium dihydrogen phosphate, 10-20g dipotassium hydrogen phosphate, 0.1-5g magnesium sulfate, 0.2-2g citric acid, and 0.005-2wt% defoamer 204. All components are fully dissolved in pure water and the volume is adjusted to 1L. The pH is adjusted to 7.0, and the mixture is autoclaved at 116℃ for 30 minutes, then cooled to below 50℃. 1-10ml of trace element solution and 5-50ml of antibiotic solution are added.

[0026] 3. Preparation of fed culture medium Each liter of supplemental culture medium comprises: 1-5g glucose, 100-1000g glycerol, 3-10g yeast extract, 5-20g tryptone, 5-15g sodium chloride, 2-5g potassium dihydrogen phosphate, 10-20g dipotassium hydrogen phosphate, 0.1-5g magnesium sulfate, 0.2-2g citric acid, and 0.005-2wt% defoamer 204. All components are fully dissolved in pure water and the volume is adjusted to 1L. The pH is adjusted to 7.0, and the mixture is autoclaved at 116℃ for 30 minutes, then cooled to below 50℃. 1-10ml of trace element solution and 5-50ml of antibiotic solution are added.

[0027] 4. Preparation of expression culture medium Each liter of expression culture medium contains: 100-1000g glycerol, 3-10g yeast extract, 5-20g tryptone, 5-15g sodium chloride, 0.1-5g magnesium sulfate, 0.2-2g citric acid, 0.1-10g astragalus extract, and 0.01-10g codonopsis extract. All components are fully dissolved in pure water and the volume is adjusted to 1L. The pH is adjusted to 7.0, and the medium is autoclaved at 116℃ for 30 minutes, then cooled to below 50℃, and 5-50ml of antibiotic solution is added.

[0028] 5. The preparation method of plasmid containing the duck circovirus Cap gene is as follows: (1) The codons of the duck circovirus Cap gene were optimized according to Gallus gallus, and the optimized sequence is as follows: ; (2) The Cap gene with optimized codons was artificially synthesized, and EcoRI and XhoRI restriction sites were added to the N-terminus and C-terminus, respectively. The gene was then cloned into the pCAGGS vector. Both gene synthesis and cloning into the vector were performed by Nanjing Genscript Biotech Co., Ltd.

[0029] Example 1 A method for increasing the proportion of supercoiled plasmids includes the following steps: (1) Preparation of strains for production Recombinant Escherichia coli containing the duck circovirus Cap gene plasmid was inoculated into basal culture medium at a ratio of 2% and incubated at 37°C for 12 hours. After passing the inspection, it was used as the production strain. Each liter of basal culture medium contained the following components: 5g glucose, 8g glycerol, 5g yeast extract, 10g tryptone, 10g sodium chloride, 2.531g potassium dihydrogen phosphate, 14.178g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.5g citric acid, 0.01wt% defoamer 204, 5ml trace element solution, 20ml antibiotic solution.

[0030] Each liter of trace element solution contains: 0.4g cobalt chloride, 5g calcium chloride, 0.3g copper chloride, 0.5g boric acid, 2.5g manganese chloride, 0.4g sodium molybdate, 2.0g zinc acetate, and 12g ferric citrate.

[0031] (2) Bacterial expansion culture Add basal culture medium to the sterilized fermenter, filling the fermenter to 50% of its maximum volume. Inoculate the production strain at a ratio of 1% into the basal culture medium. Control the culture temperature at 32℃, dissolved oxygen at 30%, and pH at 7.0. During fermentation, use ammonia to adjust the pH of the solution in the fermenter. Record fermentation parameters every 2 hours and aseptically sample the bacterial solution to determine the OD. 600 value.

[0032] (3) Feeding When the OD in the fermenter 600 When the value is 25, use supplemental culture medium based on the bacterial OD value. 600 Feeding should be performed at a rate of 1 ml / OD per liter of culture volume per hour. 600 The culture temperature was controlled at 32℃, dissolved oxygen at 30%, and pH at 7.0. Ammonia was used to adjust the pH of the solution in the fermenter during fermentation. Fermentation parameters were recorded every 2 hours, and aseptic samples were taken to measure the OD of the bacterial solution. 600 value.

[0033] Each liter of supplemental culture medium contains: 2g glucose, 500g glycerol, 5g yeast extract, 10g tryptone, 10g sodium chloride, 2.531g potassium dihydrogen phosphate, 14.178g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.5g citric acid, 0.01wt% defoamer, 5ml trace element solution, and 20ml antibiotic solution.

[0034] (4) Heating When the OD in the fermenter 600When the value is 50, gradually increase the fermenter temperature to 40℃ at a rate of 2℃ / h, and continue to feed the culture medium at a rate of 1 ml / OD per liter of culture volume per hour. 600 The dissolved oxygen level was 30%, and the pH was 7.0. Ammonia was used to adjust the pH of the solution in the fermenter during fermentation. Fermentation parameters were recorded every 2 hours, and aseptic samples were taken to measure the OD of the bacterial solution. 600 value.

[0035] (5) Plasmid expression Once the fermenter reaches the predetermined temperature, stop feeding the culture medium and add expression culture medium to the tank at a rate of 2 ml / L culture volume per hour. Control the culture temperature at 40℃, dissolved oxygen at 20%, and pH at 7.0, and continue culturing for 8 hours.

[0036] Each liter of expression culture medium contains: 500g glycerol, 5g yeast extract, 10g tryptone, 10g sodium chloride, 2.531g potassium dihydrogen phosphate, 14.178g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.5g citric acid, 5g astragalus extract, 3g codonopsis extract, and 20ml antibiotic solution.

[0037] (6) Cooling and harvesting of mycelium After plasmid expression, the temperature inside the fermenter was rapidly reduced to 16°C and maintained at 16°C for another 2 hours. Simultaneously, expression medium was continuously added to the fermenter at a rate of 2 ml / L culture volume per hour. The bacterial cells were then harvested and the plasmid was purified.

[0038] Example 2 A method for increasing the proportion of supercoiled plasmids includes the following steps: (1) Preparation of strains for production Recombinant Escherichia coli containing the duck circovirus Cap gene plasmid was inoculated into basal culture medium at a ratio of 2% and incubated at 37°C for 12 hours. After passing the inspection, it was used as the production strain. Each liter of basal culture medium contained the following components: 5g glucose, 8g glycerol, 5g yeast extract, 10g tryptone, 10g sodium chloride, 2.531g potassium dihydrogen phosphate, 14.178g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.5g citric acid, 0.01wt% defoamer 204, 5ml trace element solution, 20ml antibiotic solution.

[0039] Each liter of trace element solution contains: 0.4g cobalt chloride, 5g calcium chloride, 0.3g copper chloride, 0.5g boric acid, 2.5g manganese chloride, 0.4g sodium molybdate, 2.0g zinc acetate, and 12g ferric citrate.

[0040] (2) Bacterial expansion culture Add basal culture medium to the sterilized fermenter, filling the fermenter to 50% of its maximum volume. Inoculate the production strain at a ratio of 1% into the basal culture medium. Control the culture temperature at 32℃, dissolved oxygen at 30%, and pH at 7.0. During fermentation, use ammonia to adjust the pH of the solution in the fermenter. Record fermentation parameters every 2 hours and aseptically sample the bacterial solution to determine the OD. 600 value.

[0041] (3) Feeding When the OD in the fermenter 600 When the value is 25, use supplemental culture medium based on the bacterial OD value. 600 Feeding should be performed at a rate of 1 ml / OD per liter of culture volume per hour. 600 The culture temperature was controlled at 32℃, dissolved oxygen at 30%, and pH at 7.0. Ammonia was used to adjust the pH of the solution in the fermenter during fermentation. Fermentation parameters were recorded every 2 hours, and aseptic samples were taken to measure the OD of the bacterial solution. 600 value.

[0042] Each liter of supplemental culture medium contains: 2g glucose, 500g glycerol, 5g yeast extract, 10g tryptone, 10g sodium chloride, 2.531g potassium dihydrogen phosphate, 14.178g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.5g citric acid, 0.01wt% defoamer, 5ml trace element solution, and 20ml antibiotic solution.

[0043] (4) Heating When the OD in the fermenter 600 When the value is 50, gradually increase the fermenter temperature to 40℃ at a rate of 2℃ / h, and continue to feed the culture medium at a rate of 1 ml / OD per liter of culture volume per hour. 600 The dissolved oxygen level was 30%, and the pH was 7.0. Ammonia was used to adjust the pH of the solution in the fermenter during fermentation. Fermentation parameters were recorded every 2 hours, and aseptic samples were taken to measure the OD of the bacterial solution. 600 value.

[0044] (5) Plasmid expression Once the fermenter reaches the predetermined temperature, stop feeding the culture medium and add expression culture medium to the tank at a rate of 2 ml / L culture volume per hour. Control the culture temperature at 40℃, dissolved oxygen at 20%, and pH at 7.0, and continue culturing for 8 hours.

[0045] Each liter of expression culture medium contains: 500g glycerol, 5g yeast extract, 10g tryptone, 10g sodium chloride, 2.531g potassium dihydrogen phosphate, 14.178g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.5g citric acid, 0.5g astragalus extract, 0.05g codonopsis extract, and 20ml antibiotic solution.

[0046] (6) Cooling and harvesting of mycelium After plasmid expression, the temperature inside the fermenter was rapidly reduced to 16°C and maintained at 16°C for another 2 hours. Simultaneously, expression medium was continuously added to the fermenter at a rate of 2 ml / L culture volume per hour. The bacterial cells were then harvested and the plasmid was purified.

[0047] Example 3 A method for increasing the proportion of supercoiled plasmids includes the following steps: (1) Preparation of strains for production Recombinant Escherichia coli containing the duck circovirus Cap gene plasmid was inoculated into basal culture medium at a ratio of 2% and incubated at 37°C for 12 hours. After passing the inspection, it was used as the production strain. Each liter of basal culture medium contained the following components: 5g glucose, 8g glycerol, 5g yeast extract, 10g tryptone, 10g sodium chloride, 2.531g potassium dihydrogen phosphate, 14.178g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.5g citric acid, 0.01wt% defoamer 204, 5ml trace element solution, 20ml antibiotic solution.

[0048] Each liter of trace element solution contains: 0.4g cobalt chloride, 5g calcium chloride, 0.3g copper chloride, 0.5g boric acid, 2.5g manganese chloride, 0.4g sodium molybdate, 2.0g zinc acetate, and 12g ferric citrate.

[0049] (2) Bacterial expansion culture Add basal culture medium to the sterilized fermenter, filling the fermenter to 50% of its maximum volume. Inoculate the production strain at a ratio of 1% into the basal culture medium. Control the culture temperature at 32℃, dissolved oxygen at 30%, and pH at 7.0. During fermentation, use ammonia to adjust the pH of the solution in the fermenter. Record fermentation parameters every 2 hours and aseptically sample the bacterial solution to determine the OD. 600 value.

[0050] (3) Feeding When the OD in the fermenter 600 When the value is 25, use supplemental culture medium based on the bacterial OD value. 600 Feeding should be performed at a rate of 1 ml / OD per liter of culture volume per hour. 600The culture temperature was controlled at 32℃, dissolved oxygen at 30%, and pH at 7.0. Ammonia was used to adjust the pH of the solution in the fermenter during fermentation. Fermentation parameters were recorded every 2 hours, and aseptic samples were taken to measure the OD of the bacterial solution. 600 value.

[0051] Each liter of supplemental culture medium contains: 2g glucose, 500g glycerol, 5g yeast extract, 10g tryptone, 10g sodium chloride, 2.531g potassium dihydrogen phosphate, 14.178g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.5g citric acid, 0.01wt% defoamer, 5ml trace element solution, and 20ml antibiotic solution.

[0052] (4) Heating When the OD in the fermenter 600 When the value is 50, gradually increase the fermenter temperature to 40℃ at a rate of 2℃ / h, and continue to feed the culture medium at a rate of 1 ml / OD per liter of culture volume per hour. 600 The dissolved oxygen level was 30%, and the pH was 7.0. Ammonia was used to adjust the pH of the solution in the fermenter during fermentation. Fermentation parameters were recorded every 2 hours, and aseptic samples were taken to measure the OD of the bacterial solution. 600 value.

[0053] (5) Plasmid expression Once the fermenter reaches the predetermined temperature, stop feeding the culture medium and add expression culture medium to the tank at a rate of 2 ml / L culture volume per hour. Control the culture temperature at 40℃, dissolved oxygen at 20%, and pH at 7.0, and continue culturing for 8 hours.

[0054] Each liter of expression culture medium contains: 500g glycerol, 5g yeast extract, 10g tryptone, 10g sodium chloride, 2.531g potassium dihydrogen phosphate, 14.178g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.5g citric acid, 10g astragalus extract, 5g codonopsis extract, and 20ml antibiotic solution.

[0055] (6) Cooling and harvesting of mycelium After plasmid expression, the temperature inside the fermenter was rapidly reduced to 16°C and maintained at 16°C for another 2 hours. Simultaneously, expression medium was continuously added to the fermenter at a rate of 2 ml / L culture volume per hour. The bacterial cells were then harvested and the plasmid was purified.

[0056] Comparative Example 1 The difference from Example 1 is that the expression culture medium does not contain Codonopsis pilosula extract, while the rest of the process is the same as in Example 1.

[0057] Comparative Example 2 The difference from Example 1 is that the expression culture medium does not contain Astragalus extract, while the rest of the process is the same as in Example 1.

[0058] Comparative Example 3 The difference from Example 1 is that the expression culture medium does not contain Astragalus extract and Codonopsis extract, while the rest of the process is the same as in Example 1.

[0059] Comparative Example 4 Compared with Example 1, the difference is that the heating process in step (4) and the cooling process in step (6) are missing. Also, the culture temperature is 37°C during the feeding process in step (3), and the plasmid expression process in step (5) is adjusted as follows: The OD600nm value in the fermenter was 50. After culturing for another 4 hours, the feeding of culture medium was stopped. Expression culture medium was added to the fermenter at a rate of 2 ml / L culture volume per hour. The culture temperature was controlled at 37℃, the dissolved oxygen rate at 20%, and the pH value at 7.0. The culture was continued for another 10 hours.

[0060] The remaining process is consistent with Example 1.

[0061] Comparative Example 5 Compared with Comparative Example 4, the difference is that the expression culture medium did not contain Astragalus extract and Codonopsis extract, while the rest of the process remained the same.

[0062] Test case Determination of plasmid DNA content and supercoil ratio The bacterial cells harvested from Examples 1, 2, 3, Comparative Examples 1, 2, 3, and 4 were purified using the same process. The plasmid content was measured and converted to the yield of plasmid DNA per liter of culture volume. The proportion of supercoiled plasmids in the final product was also measured. The results are shown in Table 1 and [Table data missing]. Figure 1 .

[0063] Table 1. Plasmid DNA Yield

[0064] Table 1 and Figure 1 The results showed that, compared with the comparative examples, the schemes of Examples 1, 2, and 3 described in this invention significantly improved the plasmid DNA yield and the proportion of supercoiled plasmids in the final product. Comparative Examples 1-3 also demonstrated that the Astragalus extract and Codonopsis extract added to the expression culture medium had a significant impact on the proportion of supercoiled plasmids. Furthermore, Comparative Examples 4 and 5 further illustrated that only through the specific culture method and specific culture medium combination constructed in this invention can a high supercoiled plasmid ratio be ensured.

[0065] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A production method for increasing the proportion of supercoiled plasmids, characterized in that, Includes the following steps: (1) Recombinant Escherichia coli containing the duck circovirus Cap gene plasmid is inoculated into a basic culture medium at a ratio of 1-5% and cultured to be used as a production strain; the basic culture medium includes the following components at the following concentrations: glucose 3-10 g / L, glycerol 5-10 g / L, yeast extract 3-10 g / L, tryptone 5-20 g / L, sodium chloride 5-15 g / L, potassium dihydrogen phosphate 2-5 g / L, dipotassium hydrogen phosphate 10-20 g / L, magnesium sulfate 0.1-5 g / L, citric acid 0.2-2 g / L, defoamer 0.005-2%, trace element solution 0.001-0.01%, and antibiotic solution 0.005-0.05%; (2) Inoculate the production strain into the basic culture medium at a ratio of 1-5% and continue to culture at 32-35℃; (3) Step (2) Cultivate to OD 600 When the temperature reaches 20-30°C, supplemental feeding should be performed using fed culture medium at a rate of 0.5-1.5 ml / OD per liter of culture volume per hour. 600 The culture medium was further cultured at 32-35℃. The culture medium contained the following components at the following concentrations: glucose 1-5 g / L, glycerol 100-1000 g / L, yeast extract 3-10 g / L, tryptone 5-20 g / L, sodium chloride 5-15 g / L, potassium dihydrogen phosphate 2-5 g / L, dipotassium hydrogen phosphate 10-20 g / L, magnesium sulfate 0.1-5 g / L, citric acid 0.2-2 g / L, defoamer 204 0.005-2%, trace element solution 0.001-0.01%, and antibiotic solution 0.005-0.05%. (4) Step (3) Cultivate to OD 600 When the temperature reaches 40-60°C, supplemental feeding should be performed using fed culture medium at a rate of 0.5-1.5 ml / OD per liter of culture volume per hour. 600 During the cultivation process, the temperature is increased to 38-40℃ at a rate of 1-2℃ / h; (5) After heating to the predetermined temperature in step (4), stop adding the feeding medium, and then add the expression medium at a rate of 1~2 mL / L·h, and continue culturing at 38~40℃; the expression medium includes the following components at the following concentrations: glycerol 100~1000 g / L, yeast extract 3~10 g / L, tryptone 5~20 g / L, sodium chloride 5~15 g / L, magnesium sulfate 0.1~5 g / L, citric acid 0.2~2 g / L, astragalus extract 0.1~10 g / L, codonopsis extract 0.01~10 g / L, and antibiotic solution 0.005~0.05%; (6) After the plasmid expression in step (5) is completed, the temperature is lowered to 15~18℃, and then the expression medium is added at a rate of 3~5mL / L·h to culture for 2~5h to obtain a plasmid with a high supercoil ratio.

2. The production method according to claim 1, characterized in that, In step (1), the culture temperature is 35~37℃ and the culture time is 10~16h.

3. The production method according to claim 1, characterized in that, In step (2), the inoculation ratio of the production strain is 1-2%, the culture temperature is 32-35℃, the pH is 7.0-7.5, and the dissolved oxygen rate is 30-35%.

4. The production method according to claim 1, characterized in that, Step (3) The feeding rate is 0.5~1.0 ml / OD. 600 The culture temperature is 32~35℃, the pH is 7.0~7.5, and the dissolved oxygen rate is 30~35%.

5. The production method according to claim 1, characterized in that, Step (4) The feeding rate is 0.5~1.0 ml / OD. 600 During the cultivation process, the temperature was increased to 40℃ at a rate of 2℃ / h, the pH of the cultivation system was 7.0~7.5, and the dissolved oxygen rate was 30~35%.

6. The production method according to claim 1, characterized in that, The feeding rate in step (5) is 2 mL / L·h, the culture temperature is 40℃, the pH is 7.0~7.5, and the dissolved oxygen rate is 30~35%.

7. The production method according to claim 1, characterized in that, The trace element solutions in basal and supplemental culture media contain the following components at the following concentrations: Cobalt chloride 0.1~0.4 g / L, calcium chloride 2~5 g / L, copper chloride 0.05~0.3 g / L, boric acid 0.2~0.5 g / L, manganese chloride 0.8~2.5 g / L, sodium molybdate 0.1~0.4 g / L, zinc acetate 0.5~2.0 g / L, ferric citrate 5~12 g / L.

8. The production method according to claim 1, characterized in that, The antibiotic solution in the basal culture medium, feed culture medium, and expression culture medium was an ampicillin solution with a concentration of 100-120 g / L.

9. A plasmid with a high supercoil ratio, characterized in that, It is prepared by the method described in any one of claims 1 to 8.

10. Use of the plasmid of claim 9 in the development of DNA nucleic acid vaccines.

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