Method for producing virus packaging plasmid through escherichia coli fermentation
By redesigning the culture medium and fermentation temperature, the yield and quality of virus packaging plasmids produced by E. coli fermentation is improved, and the problems of low yield, high cost and poor plasmid quality in the existing technology are solved, achieving more efficient and safe plasmid production.
Patent Information
- Application Number
- CN202411593405.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-08
- Publication Date
- 2025-03-18
AI Technical Summary
In the prior art, E. coli fermentation of virus packaging plasmids is not high, the production cost is high, the amount of culture medium is used, tryptone with animal-derived ingredients brings biosafety risks, the quality of the plasmid is low, and the proportion of open ring plasmids is high.
By redesigning the composition and content of the culture medium and designing the fermentation temperatures at different stages, the plasmid was constructed, transferred into E. coli, and undergoing two-stage fermentation. The first stage is used to culture the medium at low temperature; the second stage is used to culture the medium at high temperature to improve the yield and quality of plasmids.
The yield and quality of virus packaging plasmids have been significantly improved, the proportion of open ring plasmids has been reduced to less than 5%, the use of culture medium has been reduced, the use of animal-derived ingredients has been avoided, the production cost has been reduced, and the accessibility of gene therapy drugs has been improved.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for producing virus packaging plasmids through Escherichia coli fermentation. Background Art
[0002] Adeno-associated virus is an important vector for gene therapy, through which the target gene can be integrated into the host gene. At present, all approved gene therapy drugs use AAV as a vector. AAV can infect a variety of cells. When the rep gene product is present, the viral DNA can easily integrate into a specific position of human chromosome 19, and it is very safe. The main technology of AAV virus at this stage is to produce a large number of packaging plasmids, and then transfect the plasmids into 293 cells to assemble into AAV viruses with target therapeutic gene fragments. The insect baculovirus protein expression system has been applied in the pharmaceutical and biotechnology industries, such as the production of vaccines, recombinant proteins and gene therapy drugs. By mass-producing plasmids of recombinant insect baculovirus DNA and infecting SF9 cells, insect baculovirus with target gene fragments can be assembled, and then a large number of recombinant proteins and gene drugs can be obtained by infecting SF9 cells.
[0003] The existing technology also mainly produces plasmids through Escherichia coli fermentation, but the existing virus-packaged plasmid fermentation process has low plasmid yield, high production cost, large amount of culture medium used, animal-derived trypsin peptone brings biosafety risks, low plasmid quality, and open-circular plasmid can reach 10% to 20%.
[0004] Virus packaging plasmid is a very important part of virus production. Taking adeno-associated virus as an example, its production cost accounts for more than 50% of adeno-associated virus gene therapy drugs. For example, the pAAV2-8 plasmid itself has a relatively long base sequence and is not compatible with E. coli, resulting in poor plasmid stability; insufficient stability will cause the plasmid to be easily degraded during fermentation, and the yield of the plasmid after degradation is not high. Moreover, the degradation products are similar to good plasmids and are difficult to separate, resulting in poor quality of the final product. It is necessary to find a method to increase the yield of virus packaging plasmids. Summary of the invention
[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a method for producing virus packaging plasmids by fermentation of Escherichia coli. The method improves the fermentation yield and reduces the amount of culture medium used by redesigning the composition and content of the culture medium and designing the fermentation temperature at different stages, and avoids trypsin from animal sources, thereby greatly improving the yield and quality of the virus packaging plasmids.
[0006] In order to solve the above technical problems, the present invention provides a method for producing virus packaging plasmids by fermentation of Escherichia coli, the method comprising: constructing a plasmid, and transferring the plasmid into Escherichia coli; culturing the transferred Escherichia coli with a culture medium, wherein the culture medium comprises yeast extract and glycerol;
[0007] The E. coli culture includes a first stage and a second stage;
[0008] During the second stage of cultivation of Escherichia coli, the culture temperature T2 ≥ 40°C for at least 5 hours.
[0009] Based on a general technical concept, the present invention also provides a method for producing a virus packaging plasmid by fermenting Escherichia coli, the method comprising: constructing a plasmid, and transferring the plasmid into Escherichia coli; culturing the transferred Escherichia coli in a culture medium, wherein the culture medium comprises yeast extract and glycerol;
[0010] The E. coli culture includes a first stage and a second stage;
[0011] The culture temperature of E. coli in the first stage is T1≤28°C;
[0012] The second stage of culturing Escherichia coli is carried out at a culture temperature T2 ≥ 40°C.
[0013] In the above method, further, the Escherichia coli culture comprises:
[0014] The culture temperature of E. coli in the first stage is 25°C≤T1≤28°C;
[0015] The culture temperature of Escherichia coli in the second stage is 40°C≤T2≤43°C.
[0016] In the above method, further, the Escherichia coli culture comprises:
[0017] The first stage of culturing of Escherichia coli is carried out using a basic culture medium, wherein the components of the basic culture medium include: 3-5 g / L yeast extract, 10-14 g / L glycerol, 2-4 g / L ammonium sulfate, 7-9 g / L dipotassium hydrogen phosphate, 1-3 g / L sodium dihydrogen phosphate, 1-3 g / L magnesium sulfate and 2-4 ml / L trace element solution.
[0018] In the above method, further, the trace element solution comprises: 10 g / L ferrous sulfate heptahydrate, 4 g / L calcium chloride dihydrate, 6 g / L anhydrous manganese sulfate, 6 g / L zinc sulfate heptahydrate, 2 g / L cobalt chloride hexahydrate, 3 g / L copper sulfate pentahydrate, 2 g / L sodium molybdate dihydrate, 1.5 g / L boric acid and 0.6 mol / L sulfuric acid.
[0019] In the above method, further, the Escherichia coli culture comprises:
[0020] The Escherichia coli was cultured in the second stage using a feed medium, wherein the feed medium components included 600-900 g / L glycerol and 30-50 g / L yeast extract.
[0021] The above method, further, when the fermentation time reaches 7 to 10 hours, feed medium is added to culture the Escherichia coli;
[0022] Preferably, the flow rate of the feed medium is: Vn=A0×(1+0.07)n;
[0023] Where n is the number of hours, Vn is the flow rate in the Nth hour, and A0 is the initial feed rate;
[0024] Preferably, the initial feeding rate is 1.6≤A0≤2.8.
[0025] In the above method, further, when the fermentation time reaches 23 to 25 hours, the flow rate of the feed medium is stopped, and the feed is continued at the flow rate at the stopping time.
[0026] In the above method, further, during the culture process, ammonia water with a concentration of less than 20% (w / v) is used to adjust the pH.
[0027] In the above method, further, the first stage culture time of Escherichia coli is 35h-45h;
[0028] In the above method, further, the Escherichia coli is a DH5α strain or a DH10Bac strain;
[0029] The second stage culture time of E. coli is 5h-15h.
[0030] Compared with the prior art, the advantages of the present invention are: (1) The present invention provides a method for producing virus packaging plasmids by fermenting Escherichia coli, by controlling the growth rate of the bacteria by combining the culture temperature and the culture medium, thereby reducing the loss of plasmids; in the late fermentation stage, the temperature is higher than the optimal growth temperature of 37°C, causing the Escherichia coli to enter a stress state, produce more plasmids, and reduce the open plasmids to less than 5%, which is much lower than the result of the conventional process where the open plasmid ratio is more than 10%.
[0031] The present invention overcomes the defects of low yield and poor quality of traditional methods, and has made substantial improvements and innovations in the method of high-density fermentation to produce virus-packaging plasmids. The culture medium and culture process used, the final plasmid expression amount is 3 to 4 times higher than that of the general fermentation method, and the proportion of open-circular plasmids is reduced to less than 5%, which can reduce the plasmid production cost by at least 30%, thereby reducing the cost of gene therapy drugs and improving the accessibility of drugs.
[0032] (2) The present invention provides a method for producing virus packaging plasmids by fermentation of E. coli. The culture medium reduces the use of yeast extract and does not use trypsin, avoids the use of raw materials of animal-derived components, and improves the safety of the product. Compared with TB culture medium, 11.8 g / L trypsin and 23.6 g / L yeast extract can save 236 g of trypsin and 392 g of yeast extract according to 20 L culture medium. The increase in yield is equivalent to the yield of 3-4 batches fermented by other fermentation methods, which saves production time and greatly saves production costs.
[0033] (3) The present invention provides a method for producing virus packaging plasmids by fermentation of Escherichia coli. In the first stage, the nutrient components in the basic culture medium are maintained at a low level, and low-temperature culture is adopted. The low nutrient and low-temperature culture is used to increase the plasmid yield and reduce the plasmid opening ratio. The second stage culture rehydration flow rate is adjusted to achieve a high mass content while maintaining the plasmid opening ratio of the less stable plasmid at a low level. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.
[0035] Figure 1 In Experiment 4 of the present invention, the fermentation temperature in the first stage was controlled at 28°C, and the fermentation temperature in the second stage was adjusted to 37°C to 43°C, and the detection results of OD600 and plasmid content were obtained.
[0036] Figure 2 In Experiment 4 of the present invention, the fermentation temperature in the second stage was controlled to be 41°C, and the fermentation temperature in the first stage was adjusted to 24°C to 30°C, and the detection results of OD600 and plasmid content were obtained. DETAILED DESCRIPTION
[0037] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby.
[0038] The materials, reagents and instruments used in the following examples can all be purchased from commercial channels. The experimental methods in the following examples are all conventional methods in the art unless otherwise specified.
[0039] Example 1
[0040] A method for producing an adeno-associated virus packaging plasmid by fermentation with Escherichia coli. The plasmid used in this embodiment is pAAV2-8 plasmid. The specific method comprises the following steps:
[0041] (1) Preparation before fermentation:
[0042] 1.1、Turbidity probe installed on the tank (OD can be calculated based on turbidity) 600 ), DO electrode and pH electrode are calibrated offline to 4.01 and 7.0, then installed into the tank, and the pipeline connections are checked for correctness.
[0043] 1.2. Add 20 L of basal culture medium (the ingredients of the basal culture medium are: 4 g / L yeast extract, 12 g / L glycerol, 3 g / L ammonium sulfate, 8 g / L dipotassium hydrogen phosphate, 2 g / L sodium dihydrogen phosphate, 2 g / L magnesium sulfate, 3 ml / L trace element solution (the trace element solution includes: 10 g / L ferrous sulfate heptahydrate, 4 g / L calcium chloride dihydrate, 6 g / L anhydrous manganese sulfate, 6 g / L zinc sulfate heptahydrate, 2 g / L cobalt chloride hexahydrate, 3 g / L copper sulfate pentahydrate, 2 g / L sodium molybdate dihydrate, 1.5 g / L boric acid and 0.6 mol / L sulfuric acid) and a defoamer with a volume fraction of 0.015% into a 50 L fermenter.
[0044] 1.3. Start the FSIP program of the tank body and sterilize it at 121℃ for 20 minutes. After the sterilization is completed, ventilate with 20L / min compressed air, start the tank pressure control program, and keep the pressure in the tank 0.5bar higher than the atmospheric pressure.
[0045] 1.4. Turn on the tank temperature control program and set the temperature to 28°C and stir at 50 rpm.
[0046] 1.5. The tank body is connected to a sterilized 2000 ml 10% (w / v) ammonia water feeding bottle (ammonia water is mainly used to adjust the pH. As the fermentation proceeds, Escherichia coli will continuously produce a large amount of acidic substances to lower the pH. In order to keep the pH within the control range, after setting a specified pH on the fermentation tank, the fermentation system can automatically add ammonia water to maintain the specified pH. In this embodiment, low-concentration ammonia water is used for feeding) and 10 L of feed medium (the components of the feed medium are 800 g / L glycerol and 40 g / L yeast extract) and wait for fermentation.
[0047] (2) Inoculation process before fermentation:
[0048] 2.1. 3 L of E. coli DH5α strain seed solution (OD 600 =8±1) Pour the bacterial solution into the sterilized feed bottle in the biosafety cabinet.
[0049] 2.2. Set the fermentation tank parameters before inoculation: link the dissolved oxygen content with the rotation speed, and maintain the dissolved oxygen above 20% by adjusting the rotation speed. The speed adjustment range is 50rpm~700rpm; temperature 28.0℃, ventilation 20L / min; pH 7.0.
[0050] 2.3. After the parameters are stable, the dissolved oxygen 100% calibration can be performed.
[0051] 2.4. Then, the secondary seed liquid of pAAV2-8 plasmid strain is added into the tank through the pipeline for inoculation, and the inoculation amount is 15% (volume fraction); the total volume of the basic culture medium is used as the benchmark; then the fermentation batch number is entered and the timed fermentation is started.
[0052] (3) Fermentation process culture:
[0053] 3.1 First stage of cultivation: The fermentation temperature was controlled at about 28°C. Feeding was started when the fermentation timer reached 7h. The feed flow rate was calculated according to the following formula:
[0054] v n =2.2×(1+0.07) n , where n is the number of hours, v n is the flow rate at the Nth hour.
[0055] Therefore, the initial flow rate of the feed medium was 2.2 g / L / h (effective concentration), and then increased by 7% every hour on the basis of 2.2 g / L / h until the fermentation timer reached 24 hours and then stopped increasing. Feeding continued at the maximum flow rate until the tank was lowered, with a total of 6040 g of feed.
[0056] 3.2. Second stage of cultivation: starting from 40h, the temperature was raised to 41°C for a total of 48h.
[0057] 3.3. Take samples at fixed points during the fermentation process and use a small plasmid extraction kit to extract the plasmid and detect the plasmid content.
[0058] Comparative Example 1
[0059] According to the method of Example 1, the basic culture medium was changed to TB culture medium (its ingredients included: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and the remaining steps were consistent with Example 1.
[0060] Comparative Example 2
[0061] According to the method of Example 1, the basic culture medium was changed to LB culture medium (its ingredients included: 11.8 g / L tryptone, 23.6 g / L yeast extract, 2.2 g / L potassium dihydrogen phosphate, 9.4 g / L potassium hydrogen phosphate, 4 ml / L glycerol), and the remaining steps were consistent with Example 1.
[0062] Experiment 1: Detection of plasmid quality and open-circle ratio.
[0063] The plasmids of Example 1, Comparative Example 1 and Comparative Example 2 were extracted using a plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., centrifugal column type) to detect the plasmid content (please add anhydrous ethanol to the rinse solution PW before use, please refer to the label on the bottle for the added volume), and the open-loop ratio was detected.
[0064] The specific steps include:
[0065] 1. Column equilibration step: add 500 μL of equilibration solution BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
[0066] 2. Dilute the fermentation solution 100 times, take 1 ml, add it to a centrifuge tube, use a regular desktop centrifuge, centrifuge at 12,000 rpm for 1 min, and remove the supernatant as much as possible.
[0067] 3. Add 250 μL of solution P1 to the centrifuge tube containing the bacterial pellet (please check whether RNase A has been added first) and use a pipette or vortex oscillator to thoroughly suspend the bacterial pellet.
[0068] 4. Add 250 μL of solution P2 to the centrifuge tube and gently flip it upside down 6-8 times to fully lyse the bacteria. Note: Mix gently and do not shake violently to avoid interrupting the genomic DNA and causing the extracted plasmid to be mixed with genomic DNA fragments. At this time, the bacterial solution should become clear and viscous. The time used should not exceed 5 minutes to avoid damage to the plasmid. If it does not become clear, it may be due to excessive bacteria and incomplete lysis. The amount of bacteria should be reduced.
[0069] 5. Add 350 μL of solution P3 to the centrifuge tube and gently turn it upside down 6-8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 minutes. Note: P3 should be mixed immediately after addition to avoid local precipitation.
[0070] 6. Use a pipette to transfer the supernatant collected in the previous step to the adsorption column CP3 (the adsorption column is placed in the collection tube). Be careful not to suck out the precipitate. Centrifuge at 12,000 rpm for 30-60 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube.
[0071] 7. Optional step: Add 500 μL of deproteinized solution PD to the adsorption column CP3, centrifuge at 12,000 rpm for 30-60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CP3 back into the collection tube.
[0072] 8. Add 600 μL of rinse solution PW to the adsorption column CP3, centrifuge at 12,000 rpm for 30-60 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube.
[0073] 9. Repeat step 8.
[0074] 10. Place the adsorption column CP3 in the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove the residual rinse solution in the adsorption column. To ensure that downstream experiments are not affected by residual ethanol, it is recommended to open the adsorption column CP3 and place it at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.
[0075] 11. Place the adsorption column CP3 in a clean centrifuge tube, add 50-100 μL of elution buffer EB to the middle part of the adsorption membrane, place at room temperature for 2 minutes, centrifuge at 12,000 rpm for 2 minutes to collect the plasmid solution in a centrifuge tube. The DNA product is stored at -20°C to prevent DNA degradation. In order to increase the recovery rate of the plasmid, the obtained solution can be added back to the adsorption column, placed at room temperature for 2 minutes, centrifuged at 12,000 rpm for 2 minutes, and the plasmid solution can be collected in a centrifuge tube.
[0076] 12. Use the nucleic acid detection mode of SH-Nano ONE ultra-micro spectrophotometer to detect the concentration of plasmid.
[0077] The ring-opening ratio was calculated by electrophoresis and gray value. The test results are listed in Table 1.
[0078] Table 1: Results of investigation of plasmid content and plasmid quality in fermentation broth in Example 1, Comparative Examples 1 and 2
[0079]
[0080] From the results in Table 1, it can be seen that after the present invention uses a medium with low yeast extract content and high glycerol content to culture Escherichia coli, the yield of the plasmid is significantly increased, and the opening ratio is also significantly lower.
[0081] Experiment 2: Investigate the fermentation content test results of the three batches in Example 1.
[0082] Three batches were produced according to the method of Example 1, and the OD600 and plasmid content at the end of fermentation were investigated. The investigation results are shown in Table 2.
[0083] Table 2: Fermentation content test results of three batches in Example 1
[0084]
[0085]
[0086] From the results in Table 2, it can be seen that the yield and quality of the pAAV2-8 plasmid can be greatly improved according to the method of Example 1.
[0087] Experiment 3: Investigate the effects of different fermentation temperatures on plasmid content and the proportion of open-circular plasmids in the first stage.
[0088] According to the method of Example 1, the fermentation temperature was adjusted to 25°C to 37°C during the first stage (0 to 40 h), and the fermentation temperature was controlled to 41°C during the second stage (40 h to 48 h). The effects of different fermentation temperatures on the plasmid content and the proportion of open-circular plasmids during the first stage were investigated. The results are shown in Table 3.
[0089] Table 3: Effects of different fermentation temperatures on plasmid content and ratio of open-circular plasmids in the first stage
[0090] From the results in Table 3, it can be seen that: when the fermentation temperature in the first stage is lower than 28°C, the proportion of open plasmids gradually decreases as the temperature decreases; when the temperature is higher than 28°C, the proportion of open plasmids increases significantly as the temperature rises.
[0091] Experiment 4: Investigate the effects of different feeding rates on plasmid content and the proportion of open-circular plasmids.
[0092] According to the method of Example 1, the feeding rate was adjusted to investigate the effects of different feeding rates on the plasmid content and the proportion of open-circular plasmids. The results are shown in Table 4.
[0093] Table 4: Effects of different feeding rates on plasmid content and ratio of open-loop plasmids
[0094] Fermentation temperature (℃) Plasmid content (mg / L) Percentage of open plasmids (%) 25 533 4.4 28 1086 4.7 31 932 8.8 34 877 11.0 37 654 13.7
[0095] From the results in Table 4, it can be seen that the feeding rate has a great influence on the plasmid content and the proportion of open-circular plasmids. When the feeding rate is 2.2 g / L / h, the yield and quality of the pAAV2-8 plasmid are greatly improved.
[0096] Experiment 5: Investigate the effects of the first-stage fermentation temperature and the second-stage fermentation temperature on OD600 and plasmid content.
[0097] According to the method of Example 1, the fermentation temperature was controlled at 24°C to 30°C in the first stage for 0 to 40 hours, and at 37°C to 43°C in the second stage for 40 to 48 hours. The effects of temperature changes in the two fermentation stages on OD600 and plasmid content were investigated. The results are shown in Table 1. Figure 1 and Figure 2 .
[0098] Initial feeding rate (g / L / h) Plasmid content (mg / L) Percentage of open plasmids (%) 1.6 758 4.1 2.2 1038 4.8 2.8 935 6.1 3.4 803 8.7 4.0 787 10.3
[0099] Figure 1The fermentation temperature was controlled at 28°C in the first stage and adjusted to 37°C to 43°C in the second stage, and the detection results of OD600 and plasmid content. Figure 2 The fermentation temperature was controlled at 41°C in the second stage, and the fermentation temperature in the first stage was adjusted to 24°C to 30°C. The test results of OD600 and plasmid content were obtained. Figure 1 and Figure 2 The results show that when the first-stage fermentation temperature is 28°C and the second-stage fermentation temperature is 41°C, the yield of pAAV2-8 plasmid is significantly higher than that of other combinations of fermentation temperatures.
[0100] Experiment 6: Fermentation results of other strains and plasmids.
[0101] According to the method of Example 1, the Escherichia coli was replaced with the DH10Bac strain Escherichia coli transformed with the recombinant insect bacmid plasmid.
[0102] The test results of three batches of fermentation content are as follows:
[0103] Fermentation batch At the end of fermentation, OD600 Plasmid content mg / L at the end of fermentation Batch 1 215 983 Batch 2 218 998 Batch 3 206 965
[0104] Experiment 7: Effect of temperature on fermentation results of other strains and plasmids.
[0105] According to the method in Example 1, the fermentation temperature in the second stage was lowered to 38°C. The fermentation results are shown in the following table:
[0106] Fermentation batch At the end of fermentation, OD600 Plasmid content mg / L at the end of fermentation Batch 1 230 906 Batch 2 222 916
[0107] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.
Claims
1. A method for producing a virus packaging plasmid by fermentation of Escherichia coli, characterized in that: The method comprises: constructing a plasmid, and transferring the plasmid into Escherichia coli; culturing the transferred Escherichia coli with a culture medium, wherein the culture medium comprises yeast extract and glycerol; The culturing of Escherichia coli includes a first stage and a second stage; During the second stage of cultivation of Escherichia coli, the culture temperature T2 ≥ 40°C for at least 5 hours.
2. A method for producing virus packaging plasmids by fermentation of Escherichia coli, characterized in that: The method comprises: constructing a plasmid, and transferring the plasmid into Escherichia coli; culturing the transferred Escherichia coli with a culture medium, wherein the culture medium comprises yeast extract and glycerol; The culturing of Escherichia coli includes a first stage and a second stage; The temperature T1 of culturing Escherichia coli in the first stage is ≤28°C; The temperature T2 for culturing Escherichia coli in the second stage is ≥40°C.
3. The method according to claim 1 or 2, characterized in that: The culturing of the Escherichia coli comprises: The temperature of culturing E. coli in the first stage is 25°C≤T1≤28°C; The temperature for culturing Escherichia coli in the second stage is 40°C≤T2≤43°C.
4. The method according to claim 1 or 2, characterized in that: The culturing of the Escherichia coli comprises: The first stage of culturing of Escherichia coli is carried out using a basic culture medium, wherein the components of the basic culture medium include: 3-5 g / L yeast extract, 10-14 g / L glycerol, 2-4 g / L ammonium sulfate, 7-9 g / L dipotassium hydrogen phosphate, 1-3 g / L sodium dihydrogen phosphate, 1-3 g / L magnesium sulfate and 2-4 ml / L trace element solution.
5. The method according to claim 4, characterized in that The trace element solution includes: 10 g / L ferrous sulfate heptahydrate, 4 g / L calcium chloride dihydrate, 6 g / L anhydrous manganese sulfate, 6 g / L zinc sulfate heptahydrate, 2 g / L cobalt chloride hexahydrate, 3 g / L copper sulfate pentahydrate, 2 g / L sodium molybdate dihydrate, 1.5 g / L boric acid and 0.6 mol / L sulfuric acid.
6. The method according to claim 1 or 2, characterized in that: The culturing of the Escherichia coli comprises: During the culture process, ammonia water with a concentration of less than 20% (w / v) is used to adjust the pH; Preferably, the Escherichia coli is cultured in the second stage using a feed medium, wherein the feed medium components include 600-900 g / L glycerol and 30-50 g / L yeast extract.
7. The method according to claim 6, characterized in that When the fermentation time reaches 7 to 10 hours, feed medium is added to culture Escherichia coli; Preferably, the flow rate of the feed medium is: V n =A0×(1+0.07) n ; Where n is the number of hours, V n is the flow rate at the Nth hour, A0 is the initial feed rate; Preferably, the initial feeding rate is 1.6≤A0≤2.8; When the fermentation time reaches 23-25 hours, stop increasing the flow rate of the feed medium and continue feeding at the flow rate at the time of stopping.
8. The method according to claim 1 or 2, characterized in that: The Escherichia coli is a DH5α strain.
9. The method according to claim 1 or 2, characterized in that: The Escherichia coli is a DH10Bac strain.
10. The method according to claim 1 or 2, characterized in that: The first stage of E. coli culture time is 35h-45h; The second stage culture time of E. coli is 5h-15h.