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Industrial purification method of plasmid DNA and plasmid DNA

A purification method and plasmid technology, applied in the field of biopharmaceuticals, can solve the problems of removal, the total recovery rate of the plasmid and the control effect of endotoxin content, the restriction of industrial purification of plasmid DNA, the impact of operators and the environment, etc., to meet the needs of the industrial industry. Chemical purification production requirements, reduce the impact of operators and the environment, the effect of simple operation

Active Publication Date: 2021-02-23
SHENZHEN WEIGUANG BIOLOGICAL PROD
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  • Claims
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AI Technical Summary

Problems solved by technology

[0003] In the prior art, the clarified filtrate after alkaline lysis still contains a large amount of host protein, which has a significant impact on the subsequent removal of RNA, the total recovery rate of plasmids, and the control of endotoxin content
The industrial purification of plasmid DNA has brought great restrictions
In the Chinese invention patent ZL200610016880.7, calcium chloride or ammonium sulfate is used for salting out and precipitation, and the filter membrane is clarified to separate the precipitate. Calcium chloride precipitates a large amount of plasmid DNA while precipitating the host protein, resulting in low recovery rate, while sulfuric acid Ammonium consumption is large, which has a great impact on operators and the environment

Method used

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  • Industrial purification method of plasmid DNA and plasmid DNA
  • Industrial purification method of plasmid DNA and plasmid DNA
  • Industrial purification method of plasmid DNA and plasmid DNA

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1 Industrial purification of DNA containing the new crown N protein particle

[0044] This example shows an industrial purification method for DNA containing the new crown N protein, which specifically includes the following steps:

[0045] (11) 2000g of Escherichia coli wet bacteria containing the new crown N protein DNA, lysed and neutralized according to the method described in the molecular cloning guide to obtain a mixture;

[0046] (12) After preliminary clarification of the above mixed solution with a tubular centrifuge, continue to filter with a combination of 5.0 μm and 0.5 μm filter elements, and rinse the filter element with purified water to obtain 58.5 L of alkali cracking filter clarification liquid;

[0047] (13) Use an ultrafiltration membrane bag with a pore size of 300KD to perform ultrafiltration and concentration on the alkali lysis filtered clarified liquid, and then use 10mmol / L trishydroxyaminomethane, 1mmol / L disodium edetate, pH8.0 The bu...

Embodiment 2

[0053] Example 2 Industrial purification of DNA containing the new crown S protein particle

[0054] This example shows a method for industrial purification of particle DNA containing the new crown S protein, which specifically includes the following steps:

[0055] (21) 2000g of Escherichia coli wet bacteria containing the new crown S protein particle DNA, the mixed solution of lysing and neutralizing according to the method described in the molecular cloning guide;

[0056] (22) After preliminary clarification with a tubular centrifuge, continue to filter with a combination of 5.0 μm and 0.5 μm filter elements, and rinse the filter element with purified water to obtain 59 L of alkali cracking filter clarification liquid;

[0057] (23) Use an ultrafiltration membrane bag with a pore size of 300KD to concentrate the clarified solution of the alkali lysis filter, and then use a buffer containing 10mmol / L trishydroxyaminomethane, 1mmol / L disodium edetate, and pH8.0 The liquid w...

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Abstract

The invention discloses an industrial purification method of plasmid DNA and plasmid DNA. The purification method comprises the following steps of, adding sodium caprylate into a plasmid DNA ultrafiltration concentrated solution, uniformly mixing, standing at normal temperature for a period of time, then standing at low temperature for a certain period of time, and then clarifying; and then sequentially carrying out ion exchange chromatography, hydrophobic chromatography, ultrafiltration desalination concentration and filtration sterilization to finish purification. According to the purification method, the sodium caprylate is applied to purification of plasmid DNA products under the low-temperature condition, operation is easy, convenient, amplifiable, operation steps are reduced, meanwhile, the total recovery rate of purified plasmids is increased, and the medicinal standard is reached; in addition, the use of ammonium sulfate is greatly reduced, and the influence on operators and environment is reduced; and the purification method is good in purification effect, the use of an ammonium sulfate reagent is greatly reduced in the purification process, the repeatability is high, theoperation is simple, and the industrial purification production requirements can be met.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to an industrial purification method of plasmid DNA and the plasmid DNA. Background technique [0002] At present, the new crown epidemic is intensifying in the world, mainly because of its strong contagiousness and repeated epidemics, which are difficult to control. At present, we mainly rely on travel control and isolation treatment to control the spread and outbreak of the epidemic, but this is not a long-term solution. Therefore, the development and application of vaccines have received extensive attention. At present, the scientific research team has laid out five technical routes of virus inactivated vaccines, nucleic acid vaccines, recombinant protein vaccines, adenovirus vector vaccines, and attenuated influenza virus vector vaccines. There are mainly adenovirus vector vaccines and inactivated vaccines that have entered the clinic. However, inactivated vaccines have a te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/101C12N15/1017
Inventor 郭土敬郭采平王雪云吴彦萍梁富黄致翔吴灼斌徐尔赞叶才文刘爱和
Owner SHENZHEN WEIGUANG BIOLOGICAL PROD
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