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Method for large-scale purification of plasmid DNA

A large-scale, plasmid-based technology, applied in the field of bioengineering, can solve the problems that plasmid DNA has not been reported in the literature, and achieve the effect of meeting the requirements of industrialized large-scale production, strong operability, and high total recovery rate

Active Publication Date: 2019-03-12
广州白云山拜迪生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no literature report on the application of diatomaceous earth as a filter aid in the field of plasmid DNA purification.

Method used

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  • Method for large-scale purification of plasmid DNA
  • Method for large-scale purification of plasmid DNA
  • Method for large-scale purification of plasmid DNA

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Experimental program
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Effect test

Embodiment 1

[0031] Weigh 20 g of each of 8 E. coli cells containing 4.8Kb plasmid DNA in parallel, and carry out alkaline lysis according to the method described in the Molecular Cloning Guide. Diatomaceous earth was added to each lysate to make the final concentration 100g / L, mixed evenly, and then the mixture was filtered through a filter with a composite pore size of 2-12 μm. Use the buffer solution containing 0mM, 20mM, 50mM, 100mM, 150mM, 200mM, 300mM and 500mM NaCl respectively to wash the diatomaceous earth and recover the plasmid DNA. Aminomethane) concentration is 100mM, pH value is 7.0. The recovery rate of plasmid DNA after filtration with diatomaceous earth was detected at different elution concentrations of sodium chloride, and the results are shown in Table 1, wherein the total content of plasmid DNA in the fermented cells was 28.3 mg.

[0032] Table 1

[0033] NaCl elution concentration (mM)

[0034] It can be seen from Table 1 that when the concentration of sod...

Embodiment 2

[0036] A kind of embodiment of the large-scale purification plasmid DNA method of the present invention, comprises the following steps:

[0037] (1) 1000g of Escherichia coli cells containing 10Kb plasmid DNA were subjected to alkali lysis by the method described in the Molecular Cloning Guide, and diatomaceous earth was added to make the final concentration 120g / L alkali lysate, and gently mixed to obtain a mixed solution;

[0038] (2) Filter the mixed solution through a 2-12 μm composite pore filter, wash the diatomaceous earth with a buffer solution containing 150 mM NaCl, 10 mM EDTA, 100 mM Tris, and a pH value of 7.0 to obtain 90.3 L of a clarified alkali lysate;

[0039] (3) Concentrate the clarified lysate through a membrane-packed ultrafiltration device. The molecular weight cut-off of the ultrafiltration membrane is 100KD, and then use a buffer solution containing 10mM EDTA, 100mM Tris, and a pH value of 7.0 to carry out displacement and diafiltration, and concentrate T...

Embodiment 3

[0055] A kind of embodiment of the large-scale purification plasmid DNA method of the present invention, comprises the following steps:

[0056] (1) 1000g of Escherichia coli cells containing 3Kb plasmid DNA were subjected to alkali lysis by the method described in the Molecular Cloning Guide, and diatomaceous earth was added to make the final concentration 50g / L alkali lysate, and mixed gently to obtain a mixed solution;

[0057] (2) Filter the mixed solution through a 2-20 μm composite pore filter, wash the diatomaceous earth with a buffer solution containing 50 mM NaCl, 100 mM EDTA, 10 mM Tris, and a pH value of 8.0 to obtain 90.1 L of a clarified alkali lysate;

[0058] (3) Concentrate the clarified lysate through a membrane-packed ultrafiltration device. The molecular weight cut-off of the ultrafiltration membrane is 50KD, and then use a buffer solution containing 10mM EDTA, 100mM Tris, and a pH value of 7.0 to carry out displacement and diafiltration, and concentrate To ...

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Abstract

The invention relates to a method for large-scale purification of plasmid DNA, and belongs to the technical field of bioengineering such as DNA vaccines, nucleic acid vaccines and gene therapy. The easy, convenient and rapid separation effect is achieved by adding diatomite into alkali lysate to aid filter and clarification, and then through purification steps such as concentrated ultrafiltration,molecular sieve chromatography, affinity chromatography, anionic exchange chromatography, replacement concentration of final products and degerming filtration of clarification lysate, plasmid DNA products meeting the medicinal standards can be produced. According to the method, the diatomite is used for the first time to aid filter and applied to the field of purification of the plasmid DNA products, and the operation method is simple, convenient and easy to enlarge; and the plasmid DNA purification method provided by the invention is high in operability and good in repeatability, the purified plasmid DNA is high in total recovery rate and meets the medicinal standards, and the requirements of mass industrial production can be met.

Description

technical field [0001] The invention relates to a method for purifying plasmid DNA on a large scale, and belongs to the technical fields of bioengineering such as DNA vaccines, nucleic acid vaccines, and gene therapy. Background technique [0002] In recent years, plasmid DNA has received more and more attention as the subject of gene therapy and nucleic acid vaccine immunity. In 1990, Wolff et al. discovered that intramuscular injection of plasmid DNA could express exogenous genes in vivo, which opened a new era of DNA immunity research. The first DNA vaccine experiment in human history conducted by Tang et al. in 1992 confirmed that the foreign gene carried by plasmid DNA and the antigenic protein synthesized in vivo can induce humoral immune response and cellular immune response. The immune characteristics of plasmid DNA products can prevent and treat diseases. Therefore, they have great application prospects in the fields of viral, bacterial, parasitic diseases, tumor i...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/11
CPCC12N15/101C12N15/11
Inventor 符美娟丘力功邱壮伟李润明倪世明
Owner 广州白云山拜迪生物医药有限公司
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