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Plasmid DNA large scale purification process

The technology of a plasmid and purification method is applied in the field of large-scale purification of plasmid DNA, which can solve the problems of medium purification effect, high cost and poor actual effect, and achieve the effect of reducing purification cost.

Active Publication Date: 2007-03-28
朱鸿飞
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AI Technical Summary

Problems solved by technology

Although affinity chromatography has no serious problems such as environmental pollution, it is time-consuming and expensive, and requires pretreatment, which limits its further use
[0008] Although the idea of ​​purifying plasmid DNA with molecular interception technology is good, the actual effect is not good. It is still in the research stage. It mainly uses the difference in molecular weight of each component in the extracted cell components to control the pore size and cross-sectional area of ​​the membrane. Membrane pressure, so as to achieve the purpose of purifying plasmid DNA
According to comprehensive reports, this technology usually requires pretreatment, many purification steps, and the purification effect is only moderate
At the same time, because plasmid DNA mainly exists in the form of elongated and negative supercoiled structure, its molecular weight is not completely correlated with the pore size of the membrane it actually needs, so the molecular retention technology cannot be fully applied to the purification of plasmid DNA

Method used

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  • Plasmid DNA large scale purification process
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  • Plasmid DNA large scale purification process

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Embodiment 1

[0033] Embodiment 1, extraction and purification of recombinant plasmid pcDNAKCpG

[0034]1. Fermentation of recombinant plasmid containing recombinant plasmid pcDNAKCpG (obtained by inserting CpG oligonucleotide in the plasmid pcDNA3.0 purchased from Invitrogen, detailed description see CN1554751A) genetically engineered recombinant bacteria (host bacteria DH5α purchased from Invitrogen): from the seed bank Take out a strain from the medium, grow into a single colony by streaking on the resistant plate, carry out the expansion culture, and inoculate it into a 10L fermentation tank at a ratio of 10%, and ferment it under the conditions of 30% dissolved oxygen and pH 7.0 Recombinant Escherichia coli containing the recombinant plasmid pcDNAKCpG, to be OD 600 When reaching 30, stop fermentation (Fig. 1), centrifuge and collect recombinant thallus, weigh 400 grams of wet bacterium.

[0035] 2. Add 5ml of solution I (25mM / L Tris-Cl, 10mM / L EDTA, 50mM / L glucose) per gram of wet bac...

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Abstract

The invention relates to a plasmid DNA large scale purification process, which comprises using 3M postassium acetate, hexadecyl trimethyl-ammonium bromide (CTAB) and TritonX-114 to purify the plasmid DNA, as a result, the host RNA, the host mixed proteins, the host enterotoxins and host genome DNA can be removed effectively.

Description

technical field [0001] The invention relates to a large-scale purification process of plasmid DNA. Background technique [0002] DNA vaccine is a new type of high-efficiency vaccine that has just been developed in recent years. Its main feature is that foreign genes are cloned into eukaryotic expression plasmid vectors. Effectively express the target protein in vivo as an antigen, so as to achieve the purpose of preventing diseases. [0003] Compared with existing vaccines, the advantages of DNA vaccines are mainly manifested in the following points: the protein antigens produced can be presented to MHC II and MHC class I molecules, and are associated with CD 4 + and CD 8 + Combined, it can cause a comprehensive immune protection response; it can produce good immune protection against intracellular parasites such as Mycobacterium tuberculosis and Leishmania; the validity period can be as long as 1 year, and it has strong immunity; the design and transformation of vaccine...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N1/21
Inventor 朱鸿飞孙怀昌张泉
Owner 朱鸿飞