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Fermentation medium for efficiently producing plasmid DNA and fermentation production method

A technology of fermentation medium and basic medium, which is applied in the field of fermentation medium for efficient production of plasmid DNA, can solve the problems of production cost impact and plasmid production, and achieve the effects of reducing production cost, shortening production cycle, and reducing volume

Pending Publication Date: 2022-03-11
BEIJING DCTY BIOTECH CO LTD
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Problems solved by technology

The composition of the medium significantly affects the yield of the plasmid, which in turn affects the production cost. Therefore, the optimization of the medium is also one of the important methods to improve the yield of the plasmid. However, there is currently no adaptation that can meet the high-density fermentation of cells and produce plasmid DNA. Media and methods

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  • Fermentation medium for efficiently producing plasmid DNA and fermentation production method
  • Fermentation medium for efficiently producing plasmid DNA and fermentation production method
  • Fermentation medium for efficiently producing plasmid DNA and fermentation production method

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preparation example Construction

[0031] The present invention also provides the preparation method of the above-mentioned fermentation medium, the preparation method of each 4000mL of the fermentation basal medium, comprising: the yeast extract, soybean peptone, glycerin, sodium chloride, magnesium sulfate heptahydrate, chloride Calcium, ammonium sulfate, thiamine, leucine and proline are mixed with water to 3700mL for sterilization, mixed with 10mL trace element solution, 200mL phosphate solution and 90mL premixed solution to obtain the fermentation basal medium; The solutes of the premix solution include thiamine, leucine and proline;

[0032] The preparation method of each 500mL of the feed medium includes mixing glycerin, yeast extract, soybean peptone, ammonium chloride, magnesium sulfate heptahydrate and water in proportion to 450mL, and sterilizing it with 50mL of 0.22μm filtered leuco Amino acid and proline mixed solution are mixed to obtain the feed medium.

[0033] The preparation method of the pre...

Embodiment 1

[0048] 1. Strain activation: take 5 μL of frozen stbl3 recombinant strain bacterial liquid (the bacteria used in this experiment are recombinant bacteria, which transform the target plasmid into stbl3 competent cells) and streak the ampicillin-resistant LB solid plate, invert Incubate overnight at 37°C in an incubator.

[0049] 2. Preparation of first-level seed liquid: Pick a single clone on the LB solid plate, inoculate it in 5 mL of ampicillin-resistant LB liquid medium, and culture it with shaking at 220 rpm at 37°C for 7.5 hours.

[0050] 3. Preparation of the secondary seed solution: Inoculate 400 μL of the primary seed solution into 400 mL of ampicillin-resistant LB liquid medium, shake at 220 rpm at 37°C for 16 hours, and use for subsequent fermentation.

[0051] 4. Fermentation pH control solution

[0052] The phosphoric acid solution was diluted to 15% (wt); the ammonia solution was diluted to a concentration of 5% (v / v), and sterilized using a 0.22 μm filter. For ...

Embodiment 2

[0083] 1. Fermentation culture

[0084] (1) Basal medium: yeast extract 48g, soybean peptone 96g, glycerin 32g, sodium chloride 16g, magnesium sulfate heptahydrate 2.5g, calcium chloride 2g, ammonium sulfate 2.5g, thiamine 2g, leucine 5g , Proline 3g. Dissolve the above ingredients in purified water, dilute to 3700mL, and autoclave at 121°C for 30 minutes;

[0085] Thiamine, leucine and proline were prepared into 90mL solution and then filtered through 0.22μm filter membrane, and then added sequentially with 200mL phosphate solution and 10mL trace element solution.

[0086] (2) Feed medium: glycerol 94g, yeast extract 56g, soybean peptone 32g, ammonium chloride 20g, leucine 8g, proline 8g, magnesium sulfate heptahydrate 2g, dilute to 450mL, 121°C, 30min autoclave;

[0087] 8 g of leucine and 8 g of proline were dissolved in 50 mL of water, filtered through a 0.22 μm filter membrane, and added to the sterilized fermentation basal medium.

[0088] (3) Fermentation process co...

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Abstract

The invention provides a fermentation medium for efficiently producing plasmid DNA and a fermentation production method, and relates to the technical field of plasmid production. The fermentation culture medium comprises a fermentation basal culture medium and a fed-batch culture medium which are independently packaged, and the combination of the fermentation basal culture medium and the fed-batch culture medium can meet the requirement of high-density fermentation and improve the yield of plasmid DNA. When the fermentation medium is used for fermentation production of Escherichia coli, the average yield of plasmids can reach 181mg / L, the volume of a bioreactor can be reduced, the purity of plasmid DNA purified in the downstream can be improved, the production cycle can be shortened, and the equipment investment can be reduced, so that the production cost is reduced, and the purpose of improving the production efficiency is achieved.

Description

technical field [0001] The invention belongs to the technical field of plasmid production, and in particular relates to a fermentation medium and a fermentation production method for efficiently producing plasmid DNA. Background technique [0002] With the further development of DNA vaccines, cell therapy and gene therapy, the demand for plasmid DNA has increased, and higher requirements have been put forward for industrial production. The production of plasmid DNA has become more and more important. Escherichia coli is used to prepare clinical-grade plasmid DNA on a large scale received more and more attention. To date, most efforts in the development of plasmid DNA production processes have focused on downstream processing. However, product quality and yield ultimately depend on the fermentation strategy. By optimizing the growth environment of recombinant bacteria, the yield of bacteria, plasmid yield and plasmid quality can be improved. Therefore, in order to be able ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/64C12P19/34C12R1/19
CPCC12N1/20C12N15/64C12P19/34
Inventor 焦顺昌张嵘张凤娇
Owner BEIJING DCTY BIOTECH CO LTD
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