Preparation method of recombinant plasmid
By transforming in E. coli competent cells and combining temperature control and glycerol fermentation, the problem of generating impurities of small plasmids in fermentation production is solved, and the preparation of high-quality plasmids is achieved, reducing purification pressure and process cycle.
Patent Information
- Application Number
- CN202411855381.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-16
- Publication Date
- 2025-03-21
AI Technical Summary
The existing recombinant plasmids are prone to produce small plasmid impurities during the fermentation and production process, which affects the transfection efficiency and IVT yield. It is difficult for existing processes to completely avoid their production, resulting in high difficulty in downstream purification and long process cycle.
The production of small plasmid impurities is reduced or avoided by transforming the recombinant plasmid into E. coli competent cells with recA1 mutations, combining temperature control and glycerol as a fermentation carbon source.
It effectively reduces the production of small plasmid impurities, reduces the development pressure of downstream purification, shortens the overall process development cycle, and obtains high-quality plasmids.
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Figure CN119662686A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for preparing a recombinant plasmid. background
[0003] With the continuous development of the biopharmaceutical industry, the research and development and application of cell and gene therapy in my country are accelerating. High-quality and high-purity plasmids are key components in the process of cell and gene therapy. They are widely used in the development of naked plasmids, DNA vaccines, mRNA and viral vectors. As front-end materials, their high quality and high purity can provide guarantees for subsequent development. Due to the presence of repeated fragments, polyA / T fragments or certain toxicity of plasmids expressing proteins in the host, the structure of plasmids in the host strain is unstable. During the fermentation and expansion process, some fragments are easily lost during the replication and synthesis of plasmids, resulting in small plasmid impurities smaller than the original plasmid. The primary structure of small plasmid impurities is different from that of the target plasmid. When the target plasmid is used as a viral packaging plasmid, small plasmid impurities will seriously affect the efficiency of transfection. When the target plasmid is used as an mRNA plasmid, small plasmid impurities will seriously affect the IVT yield and further affect the subsequent in vivo efficacy.
[0004] In the current large-scale fermentation preparation process of recombinant plasmids, the only way to avoid or minimize small plasmid impurities during fermentation production is to optimize the original plasmid backbone and the sequence of the target gene. This method has a long cycle and cannot guarantee that the optimized plasmid backbone and sequence can completely avoid the generation of small plasmid impurities. In the current production process, downstream purification is still the main method to remove small plasmid impurities, and small plasmid impurities are difficult to remove during the purification process, resulting in high development pressure for downstream purification to remove small plasmid impurities and a long cycle for the overall process development.
[0005] Therefore, developing a method that can reduce or even avoid the generation of small plasmid impurities by unstable plasmids during fermentation production will greatly reduce the development pressure of downstream purification to remove small plasmid impurities, while greatly saving the overall process development cycle. Summary of the invention
[0006] The object of the present invention is to provide a method for preparing a recombinant plasmid, which can reduce or even avoid the generation of small plasmid impurities during fermentation production.
[0007] In order to achieve the above object, the present invention provides the following technical solutions:
[0008] A method for preparing a recombinant plasmid, the method comprising the following steps:
[0009] (1) Transform the recombinant plasmid into competent E. coli cells, then culture at 25-35°C and screen for single clones;
[0010] (2) fermenting the screened single clone at 25-35° C., or first fermenting at 25-35° C. and then fermenting at 40-45° C., wherein the carbon source used in the fermentation is glycerol.
[0011] In the embodiment of the present invention, the competent E. coli cell is preferably a mutant strain having a recA1 mutation, which can reduce the occurrence of recombination of cloned DNA and thus reduce the frequency of erroneous recombination.
[0012] In the embodiment of the present invention, the E. coli competent cells are DH5α competent cells or Stable competent cells, wherein Stable competent cells include Stbl3 competent cells and Stbl2 competent cells.
[0013] Preferably, in step (1), the culture and screening of single clones are carried out at 28-32°C.
[0014] According to some specific embodiments of the present invention, in step (1), the single clones are cultured and screened at 29-31°C.
[0015] Preferably, in step (2), the screened single clone is fermented at 28-32°C, or first fermented at 28-32°C and then fermented at 40-42°C.
[0016] Furthermore, in the step (2), the screened single clone is fermented at 29-31°C, or the screened single clone is first fermented at 29-31°C and then fermented at 41-42°C.
[0017] Furthermore, in step (2), the fermentation is carried out at 28-32°C from the start of fermentation to when OD600 reaches 30; and the fermentation is carried out at 40-42°C from when OD600 is 30 to the end of fermentation.
[0018] Preferably, in step (2), the single clone is first inoculated into the fermentation substrate for fermentation culture, and then feed culture medium is continuously added for continuous fed-batch fermentation culture.
[0019] In an embodiment of the present invention, the fermentation base material formula is: 10-50 g / L glycerol, 10-30 g / L yeast extract, 10-30 g / L yeast peptone, 2-10 g / L dipotassium hydrogen phosphate dodecahydrate, 2-10 g / L dipotassium hydrogen phosphate trihydrate, 0.5-5 g / L potassium dihydrogen phosphate, 0-3 g / L sodium chloride, 0.5-3 g / L anhydrous magnesium sulfate, and 5-15 g / L ammonium sulfate.
[0020] Further preferably, the fermentation base material formula is: 30-50 g / L glycerol, 15-25 g / L yeast extract, 15-25 g / L yeast peptone, 2-8 g / L dipotassium hydrogen phosphate dodecahydrate, 2-8 g / L dipotassium hydrogen phosphate trihydrate, 1-2 g / L potassium dihydrogen phosphate, 1-2 g / L sodium chloride, 1-2 g / L anhydrous magnesium sulfate, and 8-12 g / L ammonium sulfate.
[0021] In an embodiment of the present invention, the feed medium formula is: 100-500 g / L glycerol, 100-250 g / L yeast extract, 10-30 g / L yeast peptone, 10-20 g / L ammonium sulfate, and 0.5-5 g / L sodium chloride.
[0022] Further preferably, the feed medium formula is: 350-450 g / L glycerol, 100-150 g / L yeast extract, 15-25 g / L yeast peptone, 12-18 g / L ammonium sulfate, and 1-3 g / L sodium chloride.
[0023] In some embodiments of the present invention, in step (2), the fermentation culture is carried out until OD 600 When the fermentation temperature is 15 °C, feed was started and the feed rate was 10 ml / L / h to 15 ml / L / h until the end of fermentation.
[0024] In some other embodiments of the present invention, in step (2), when OD 600 Start feeding when the temperature reaches 15:00, and continue feeding at a rate of 2-4 ml / L / h until DO recovers; from the time when dissolved oxygen recovers until fermentation reaches 18 hours, feed at a rate of 5-7 ml / L / h; from 18 hours to 24 hours of fermentation, feed at a rate of 8-10 ml / L / h; from 24 hours of fermentation to the end of fermentation, feed at a rate of 11-15 ml / L / h.
[0025] According to some specific embodiments of the present invention, step (1) is: allowing the competent E. coli cells to stand on ice to melt, adding the recombinant plasmid stock solution, shaking and mixing evenly, standing for 20 to 25 minutes, heat shocking in a 41 to 43°C water bath for 40 to 50 seconds, standing on ice for 2 to 5 minutes, adding antibiotic-free LB culture medium, incubating at 25 to 35°C, 80 to 150 rpm for 0.5 to 2 hours, taking the bacterial solution and coating it on a resistant LB agar plate, inverting and culturing at 25 to 35°C for 16 to 22 hours, selecting single clones with uniform size and shape, adding antibiotic-free LB culture medium, culturing overnight at 25 to 35°C, 200 to 250 rpm, and selecting clones with correct sequencing for fermentation.
[0026] In the embodiment of the present invention, the recombinant plasmid is a supercoiled plasmid.
[0027] The preparation method of the invention is suitable for large-scale fermentation production of supercoiled plasmids, and is also suitable for the early preparation of linear plasmids containing Poly-A.
[0028] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:
[0029] The present invention provides a process that can reduce or even avoid the generation of small plasmid impurities in structurally unstable plasmids during large-scale fermentation production. Compared with traditional transformation screening and fermentation processes, the present invention uses specific competent cells, combines temperature control and fermentation carbon source selection throughout the process, reduces or controls the early growth rate of the strain, and can reduce the generation of small plasmid impurities. It is further combined with specific culture media and fermentation condition control to avoid the generation of small plasmid impurities while reducing the impact on the plasmid expression amount, thereby obtaining high-quality plasmids, which will greatly reduce the development pressure of downstream purification to remove small plasmid impurities, and greatly save the cycle of overall process development. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 This is the gel analysis of the fermentation sample of Example 1;
[0031] Figure 2 This is the gel analysis of the fermentation sample of Comparative Example 1;
[0032] Figure 3 for Figure 2 Comparison chart of 2000bp miscellaneous band sequencing and original plasmid sequence;
[0033] Figure 4 for Figure 2 Comparison chart of 5000bp miscellaneous band sequencing and original plasmid sequence;
[0034] Figure 5 The gelatin profiles of the fermentation samples of Example 2 and Comparative Example 2 are shown;
[0035] Figure 6 The gelatin spectra of the fermentation samples of Example 3, Comparative Example 3 and Example 4 are shown. DETAILED DESCRIPTION
[0036] In order to reduce or even avoid the generation of small plasmid impurities by unstable plasmids during fermentation production, the inventors of the present application have conducted a lot of research on different stages of the fermentation preparation method, including temperature control, fermentation carbon source selection, fermentation condition control and other aspects throughout the preparation process. It was found that reducing or controlling the early growth rate of the strain can reduce the generation of small plasmid impurities. After a large number of experiments, it was confirmed that appropriately reducing the culture and fermentation temperature combined with the preferred carbon source can reduce or even avoid the generation of small plasmid impurities while reducing the impact on the plasmid expression amount.
[0037] In the embodiments of the present invention, for the Escherichia coli expression system, after appropriately lowering the culture and fermentation temperatures, the formulations of the fermentation base and feed medium during fermentation production were continuously optimized, and it was found that under the selected formulation, only using glycerol as the carbon source could further avoid the production, while when using glucose, mannitol, starch or a combination thereof, even if the culture and fermentation temperatures were appropriately lowered, some small plasmid impurities were still produced.
[0038] The technical solution of the present invention is as follows:
[0039] (1) Transform the recombinant plasmid into competent E. coli cells, then culture at 25-35°C and screen for single clones;
[0040] (2) fermenting the screened single clone at 25-35° C., or first fermenting at 25-35° C. and then fermenting at 40-45° C., wherein the carbon source used in the fermentation is glycerol.
[0041] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.
[0042] The following examples and comparative examples were respectively subjected to plasmid mini-extraction using the Tiangen Plasmid Mini-Extraction Kit with reference to its instructions. Plasmid expression level: 100 μL of fermentation broth was centrifuged to collect the bacterial cells and then the plasmid was extracted using the Tiangen Plasmid Mini-Extraction Kit. 100 μL of ultrapure water was used to elute and detect the concentration, which is the plasmid expression level (mg / L). Agarose gel electrophoresis was used for running.
[0043] In the following examples and comparative examples, the fermentation substrate and feed medium were all homemade, wherein glucose was from Sinopharm Chemical Reagent Co., Ltd., item number 63005518; glycerol was from Hunan Erkang Pharmaceutical Co., Ltd.; yeast extract was from Angel Yeast Co., Ltd., item number FM885; yeast peptone was from Angel Yeast Co., Ltd., item number FP108. Other inorganic salt components were from Sinopharm Group Co., Ltd.
[0044] Example 1
[0045] This embodiment provides a method for preparing a recombinant plasmid. The plasmid size of the plasmid stock solution used in this embodiment is 7023 bp. The preparation method of this embodiment includes the following steps:
[0046] (1) Plasmid transformation and single clone screening:
[0047] After DH5α competent cells were allowed to stand on ice and melted, 1 μL of plasmid stock solution was added, and after gently shaking and mixing, it was allowed to stand for 25 min, and then heat-shocked in a 42°C water bath for 45 s, and then allowed to stand on ice for 5 min, 800 μL of non-resistant LB medium was added, and the culture was carried out at 30°C and 100 rpm for 1 h. 40 μL of bacterial solution was applied to a resistant LB agar plate (ampicillin resistance), and the culture was inverted for 20 h. Monoclones with uniform size and shape were selected on the plate after culture, and 4 mL of resistant LB medium (ampicillin resistance) were added respectively, and cultured overnight at 30°C and 220 rpm. The bacterial solution after culture was used to carry out plasmid extraction using the Tiangen plasmid extraction kit. The plasmid extraction concentration and supercoil content were detected, and sequencing was performed. It is preferred that the extraction concentration is high, the supercoil content is high, and the clones with correct sequencing are verified on the tank (5 clones were selected for experiment in this embodiment).
[0048] (2) Fermentation production:
[0049] The five clones screened in step (1) were cultured in tanks respectively. The selected clones were inoculated into 3 L fermentation base material at a rate of 5%. The fermentation time was calculated from the time of inoculation. The whole fermentation was carried out at 30° C. The stirring speed was linked to the fermentation dissolved oxygen content. The stirring speed was adjusted within the range of 200-1200 rpm according to the fermentation dissolved oxygen content. The fermentation dissolved oxygen content was controlled at 35±10%, the pH was 6.9±0.2, and the feeding strategy was as follows: when OD 600 Feeding began at 15:00, and continued at a feeding rate of 3 ml / L / h until DO recovered; feeding was continued at a feeding rate of 6 ml / L / h from the time when dissolved oxygen recovered until fermentation reached 18 hours; feeding was continued at a feeding rate of 8 ml / L / h from 18 hours to 24 hours; feeding was continued at a feeding rate of 12 ml / L / h from 24 hours to 30 hours or until the end of fermentation. The fermentation base material formula is: 40 g / L glycerol, 20 g / L yeast extract, 20 g / L yeast peptone, 5 g / L dipotassium hydrogen phosphate dodecahydrate, 5 g / L dipotassium hydrogen phosphate trihydrate, 2 g / L potassium dihydrogen phosphate, 1.5 g / L sodium chloride, 1.5 g / L anhydrous magnesium sulfate, and 10 g / L ammonium sulfate. The feed medium formula is: 300 g / L glycerol, 130 g / L yeast extract, 20 g / L yeast peptone, 15 g / L ammonium sulfate, and 2.5 g / L sodium chloride.
[0050] After 30 hours of fermentation, samples were taken to detect the expression of plasmids in the fermentation broth. The expression of plasmids in 5 clones was between 150mg / L and 400mg / L. The fermented bacteria were subjected to plasmid mini-extraction. After the mini-extraction samples were run on the gel, the formation of mixed bands after fermentation was observed under strong contrast conditions. The results are shown in Figure 1 .
[0051] Figure 1show, Figure 1 It showed that there were no mixed bands in the plasmid stock solution, and there were no mixed bands in the fermentation microextract samples of clone 1, clone 2, clone 3, clone 4 and clone 5, and the sizes were basically consistent with the target plasmid.
[0052] Comparative Example 1
[0053] This comparative example provides another method for preparing a recombinant plasmid, which comprises the following steps:
[0054] (1) Plasmid transformation:
[0055] After thawing the DH5α competent cells on ice, add 1 μL of plasmid stock solution, gently shake to mix, let stand for 25 min, heat shock in a 42°C water bath for 45 s, let stand on ice for 5 min, add 800 μL of antibiotic-free LB medium, incubate at 37°C, 100 rpm for 1 h, take 40 μL of bacterial solution to spread on resistant LB agar plates (ampicillin resistance), and invert and culture for 20 h.
[0056] (2) Monoclonal screening:
[0057] Select single clones of uniform size and shape from the plate after culture in step (1), add them to 4 mL of resistant LB medium (ampicillin resistance), and culture them overnight at 37°C and 220 rpm. Use the Tiangen plasmid mini-extraction kit to extract plasmids from the cultured bacterial liquid. Detect the plasmid mini-extraction concentration and supercoil content, and perform sequencing. Prefer clones with high mini-extraction concentration, high supercoil content, and correct sequencing to be tested in the tank (5 clones were selected for the experiment in this comparative example)
[0058] (3) Fermentation production:
[0059] The five clones selected in step (2) were cultured in tanks respectively, and the selected clones were inoculated into 3L fermentation base material at an inoculum size of 5%. The fermentation time was calculated from the time of inoculation, and the fermentation was carried out at 37°C throughout the whole process. The stirring speed was linked to the fermentation dissolved oxygen content, and the stirring speed was adjusted within the range of 200-1200rpm according to the fermentation dissolved oxygen content. The fermentation dissolved oxygen content was controlled at 35±10%, and the pH was 6.9±0.2. The feeding strategy was: when OD 600Feeding starts at 15:00, and continues at a feeding rate of 3ml / L / h until DO recovers; feeding is carried out at a feeding rate of 6ml / L / h from the time when dissolved oxygen recovers to 18h of fermentation; feeding is carried out at a feeding rate of 8ml / L / h from 18h to 24h of fermentation; feeding is carried out at a feeding rate of 12ml / L / h from 24h to 30h of fermentation or until the end of fermentation. The fermentation base material formula is: glucose 40g / L, yeast extract 20g / L, yeast peptone 20g / L, dipotassium hydrogen phosphate dodecahydrate 5g / L, dipotassium hydrogen phosphate trihydrate 5g / L, potassium dihydrogen phosphate 2g / L, sodium chloride 1.5g / L, anhydrous magnesium sulfate 1.5g / L, ammonium sulfate 10g / L. The feed medium formula is: glucose 300g / L, yeast extract 130g / L, yeast peptone 20g / L, ammonium sulfate 15g / L, sodium chloride 2.5g / L.
[0060] After 30 hours of fermentation, samples were taken to detect the expression of plasmids in the fermentation broth. The expression of plasmids in the five clones was between 288 mg / L and 396 mg / L. The fermented bacteria were subjected to plasmid mini-extraction. After the mini-extraction samples were run on the gel, the formation of mixed bands after fermentation was observed under strong contrast conditions. The results are shown in Figure 2 .
[0061] Figure 2 The plasmid stock solution showed no miscellaneous bands, the fermentation microextract sample of clone 1 had two miscellaneous bands of about 5000bp, the fermentation microextract sample of clone 2 had one miscellaneous band of about 5000bp, the fermentation microextract sample of clone 3 had two miscellaneous bands of about 5000bp and one miscellaneous band of about 2000bp, the fermentation microextract sample of clone 4 had one miscellaneous band of about 5000bp, and the fermentation microextract sample of clone 5 had one miscellaneous band of about 5000bp.
[0062] The 2000bp and 5000bp bands in the above-mentioned small extract samples were cut and recovered, and the recovered bands were sequenced and analyzed. The bands were sequenced and compared with the original plasmid sequence (the sequence of the plasmid in the plasmid stock solution). The comparison results of the 2000bp band are shown in Figure 3 , 5000bp mixed band comparison results are shown in Figure 4. After comparing the sequencing results, the 2000bp miscellaneous band was confirmed to be a 2945-2948bp circular plasmid after NGS splicing. Its sequence is a sequence containing resistance, Ori, CMV promoter and T7 promoter elements. The missing part is the target band fragment, and the 8bp sequence before the missing fragment is "GACCCCG", which is consistent with the 8bp sequence information after the missing fragment. It can be judged that the cause of the 2000bp miscellaneous band is due to homologous recombination. After Sanger sequencing, the sequence of the 5000bp miscellaneous band is a sequence containing resistance, Ori, CMV promoter, T7 promoter and part of the target fragment. The missing part is a small amount of the target band, 3'UTR, polyA and part of the skeleton sequence. From the sequencing results of the miscellaneous band, it can be seen that the plasmid structure of the target recombinant plasmid is unstable, and it disintegrates into small plasmids during the fermentation process to form impurities.
[0063] Example 2
[0064] This example provides another method for fermentation production using clone 1 in Example 1. The fermentation process is basically the same as in Example 1, except that the feeding strategy and fermentation time during fermentation production are different. The feeding strategy in this example is: when OD600 reaches 15, feed at a feeding rate of 12 ml / L / h until fermentation is completed for 24 hours or until the fermentation is completed. After 24 hours of fermentation, the plasmid expression level in the fermentation broth was sampled and detected to be 392 mg / L. The gel run results are shown in FIG. Figure 5 Lane 3 in the sample has no debris bands.
[0065] Comparative Example 2
[0066] This comparative example provides another method for fermentation production using clone 1 in Example 1. The fermentation process is basically the same as that in Example 2, except that the carbon source in the fermentation substrate and feed medium is replaced with glucose, that is, the fermentation substrate and feed medium in Comparative Example 1 are replaced. The expression level in this comparative example is 249 mg / L, and the gel run results are shown in Figure 5 Lane 2 in the sample showed multiple miscellaneous bands between 3000 and 4000 bp.
[0067] Example 3
[0068] This example provides another method for fermentation production using clone 2 in Example 1. The fermentation process is basically the same as in Example 1, except that the feeding strategy and fermentation time during fermentation production are different. The feeding strategy in this example is: when OD600 reaches 15, feed at a feeding rate of 12 ml / L / h until fermentation is completed for 24 hours or until the fermentation is completed. After 24 hours of fermentation, the plasmid expression level in the fermentation broth was sampled and detected to be 211 mg / L. The gel run results are shown in FIG. Figure 6 Lane 2 in the sample has no debris bands.
[0069] Comparative Example 3
[0070] This example provides another method for fermentation production using clone 2 in Example 1. The fermentation process is basically the same as that in Example 3, except that the fermentation temperature is 37°C throughout the entire process. After 24 hours of fermentation, the plasmid expression level in the fermentation broth was 386 mg / L. The gel run results are shown in Figure 6 In lane 1, there is one mixed band at 2000bp and one at 3000bp.
[0071] Example 4
[0072] This example provides another method for fermentation production using clone 2 in Example 1. The fermentation process is basically the same as in Example 1, except that the feeding strategy, fermentation time and fermentation temperature during fermentation production are different. The feeding strategy in this example is: when OD600 reaches 15, start feeding at a feeding rate of 12 ml / L / h until fermentation for 24 hours or until fermentation ends. The fermentation temperature is controlled as follows: from the start of fermentation to OD600 reaching 30, fermentation is carried out at 30°C; from OD600 being 30 to fermentation for 24 hours or until fermentation ends, fermentation is carried out at 42°C. After 24 hours of fermentation, sampling was performed to detect the plasmid expression level in the fermentation broth, which was 315 mg / L. The gel running results are shown in Figure 2. Figure 6 Lane 3 in the sample has no debris bands.
[0073] Example 5
[0074] This example provides another method for preparing a recombinant plasmid with a stable plasmid structure. The method is the same as that in Example 1, except that the target plasmid is different. The plasmid stock solution used in this example has a plasmid size of 5036, and no impurity bands are found in the gel after sampling and running after 24 hours of fermentation.
[0075] The above implementation cases are only preferred examples of the present invention and are not intended to limit the protection scope of the present invention. Any modifications or improvements made without departing from the spirit of the present invention shall fall within the scope of protection required by the present invention.
Claims
1. A method for preparing a recombinant plasmid, characterized in that: The preparation method comprises the following steps: (1) Transform the recombinant plasmid into competent E. coli cells, then culture at 25-35°C and screen for single clones; (2) fermenting the screened single clone at 25-35° C., or first fermenting at 25-35° C. and then fermenting at 40-45° C., wherein the carbon source used in the fermentation is glycerol.
2. The preparation method according to claim 1, characterized in that: The E. coli competent cells are DH5α competent cells or Stable competent cells.
3. The preparation method according to claim 1, characterized in that: In the step (1), the single clones are cultured and screened at 28-32°C.
4. The preparation method according to claim 1, characterized in that: In the step (2), the screened single clone is fermented at 28-32°C, or first fermented at 28-32°C and then fermented at 40-42°C.
5. The preparation method according to claim 4, characterized in that: In the step (2), the fermentation is carried out at 28-32°C from the start of fermentation to the time when OD600 reaches 30; and the fermentation is carried out at 40-42°C from the time when OD600 reaches 30 to the end of fermentation.
6. The preparation method according to claim 1, characterized in that: In the step (2), a single clone is first inoculated into a fermentation substrate for fermentation culture, and then feed culture medium is continuously added for continuous fed-batch fermentation culture.
7. The preparation method according to claim 6, characterized in that: The fermentation base material formula is: 10-50 g / L glycerol, 10-30 g / L yeast extract, 10-30 g / L yeast peptone, 2-10 g / L dipotassium hydrogen phosphate dodecahydrate, 2-10 g / L dipotassium hydrogen phosphate trihydrate, 0.5-5 g / L potassium dihydrogen phosphate, 0-3 g / L sodium chloride, 0.5-3 g / L anhydrous magnesium sulfate, and 5-15 g / L ammonium sulfate; The feed culture medium has a formula of: 100-500 g / L glycerol, 100-250 g / L yeast extract, 10-30 g / L yeast peptone, 10-20 g / L ammonium sulfate, and 0.5-5 g / L sodium chloride.
8. The preparation method according to claim 6, characterized in that: In step (2), the fermentation culture is carried out until OD 600 When the temperature is 15°C, feed is started until the fermentation is finished, and the feed rate is 10 ml / L / h to 15 ml / L / h; Or, when OD 600 Start feeding when the temperature reaches 15:00, and continue feeding at a rate of 2-4 ml / L / h until DO recovers; from the time when dissolved oxygen recovers until fermentation reaches 18 hours, feed at a rate of 5-7 ml / L / h; from 18 hours to 24 hours of fermentation, feed at a rate of 8-10 ml / L / h; from 24 hours of fermentation to the end of fermentation, feed at a rate of 11-15 ml / L / h.
9. The preparation method according to claim 1, characterized in that: The step (1) comprises: allowing the competent E. coli cells to stand on ice to melt, adding the recombinant plasmid stock solution, oscillating and mixing evenly, standing for 20 to 25 minutes, heat-shock in a water bath at 41 to 43° C. for 40 to 50 seconds, standing on ice for 2 to 5 minutes, adding an antibiotic-free LB medium, incubating at 25 to 35° C. and 80 to 150 rpm for 0.5 to 2 hours, applying the bacterial solution on a resistant LB agar plate, incubating at 25 to 35° C. and inverting for 16 to 22 hours, selecting single clones with uniform size and shape, adding an antibiotic-free LB medium, and culturing overnight at 25 to 35° C. and 200 to 250 rpm, and selecting clones with correct sequencing for fermentation.
10. The preparation method according to claim 1, characterized in that: The recombinant plasmid is a supercoiled plasmid.