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High-purity minicircle DNA (deoxyribonucleic acid) and preparation method and application thereof

A high-purity, micro-circular technology, applied in DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as hidden dangers of clinical application of micro-circular DNA, and achieve the effect of improving safety.

Active Publication Date: 2016-02-10
SYNO MINICIRCLE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the presence of 1-3% backbone plasmids and / or parental plasmids brings hidden dangers to the clinical application of minicircle DNA

Method used

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  • High-purity minicircle DNA (deoxyribonucleic acid) and preparation method and application thereof
  • High-purity minicircle DNA (deoxyribonucleic acid) and preparation method and application thereof
  • High-purity minicircle DNA (deoxyribonucleic acid) and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0629] The construction of parental plasmids ATriplex15DsRed, Triplex15DsRed and ATriplex15DsRed, 2TTTriplex21DsRed, Triplex21DsRed with the target sequence includes the following steps:

[0630] a. Using the plasmid ZY781.CMV.DsRed.bpA as a template (the preparation method of ZY781.CMV.DsRed.bpA is: insert the CMV promoter at the SpeI and EcoRI sites of the pMC.BESPX plasmid, insert the DsRed gene at the EcoRI and SalI sites , PspOMI and ScaI sites were inserted into SV40bpA to obtain the ZY781.CMV.DsRed.bpA plasmid, wherein the gene accession numbers of the CMV promoter, DsRed gene and SV40bpA were respectively BD015377.1, FJ226077.1 and NC_001669.1) , using the following 5 pairs of primers to carry out PCR: the first pair of primers: ATriplex15-F (SEQIDNO:41) and ATriplex-R (SEQIDNO:44); the second pair of primers: Triplex15-F (SEQIDNO:21) and Triplex15-R (SEQIDNO:22); The third pair of primers: ATriplex21-F (SEQIDNO:42) and Triplex-R (SEQIDNO:44); The fourth pair of primer...

Embodiment 2

[0638] Construct parental plasmid ZY781-triplex15, ZY781-triplex21 with target sequence, comprising the following steps:

[0639] The plasmid ZY781.bpAp (the PspOMI and ScaI sites of MC.BESPX were inserted into the SV40bpA fragment to obtain the plasmid ZY781.bpAp, and the gene accession number of the SV40bpA is NC_001669.1) was used as a template; PCR was performed with the following 2 pairs of primers:

[0640] The first pair of primers: Triplex15-F (SEQ ID NO:21) and Triplex15-R (SEQ ID NO:22);

[0641] The second pair of primers: Triplex21-F (SEQ ID NO:23) and Triplex21-R (SEQ ID NO:24);

[0642] The PCR products were recovered by gel respectively, and the kan fragments with 6 Triplex15 target sequences were obtained respectively. The arrangement of the 6 Triplex15 target sequences was as follows: there were 3 tandem repeated Triplex15 target sequences in the upper and lower reaches of the Kan gene; 4 Triplex21 The kan fragment of the target sequence, wherein the four Tri...

Embodiment 3

[0645] Construction of parental plasmids Triplex21CMV.bpA, Triplex21RSV.bpA, Triplex21Ubc.bpA and Triplex21ApoE.bpA with target sequences comprises the following steps:

[0646] Using primers described in Table 1 to CMV-F (SEQIDNO:25) and CMV-R (SEQIDNO:26), RSV-F (SEQIDNO:27) and RSV-R (SEQIDNO:28), Ubc-F (SEQIDNO:28) respectively : 29) and Ubc-R (SEQIDNO: 30), ApoE-F (SEQIDNO: 31) and ApoE-R (SEQIDNO: 32) carry out PCR, amplify CMV, RSV, Ubc, ApoE promoter respectively, described PCR amplifies The genebank accession numbers of the added template genes are: CMV promoter (genebank accession number is BD015377.1), RSV promoter (genebank accession number is M77786.1), Ubc promoter (genebank accession number is NG_027722.2), ApoE promoter (genebank accession number is D38257.1); each PCR product is connected to ZY781-triplex21 through the corresponding restriction sites through conventional cloning steps, and after identification by restriction restriction and sequencing, the clo...

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Abstract

The invention provides a high-purity minicircle DNA (deoxyribonucleic acid) and a preparation method and application thereof. The preparation method includes the steps: 1) providing a parental plasmid containing a target sequence, wherein the parental plasmid has a specific recombination site, a nucleotide sequence of a skeleton DNA and a nucleotide sequence of the minicircle DNA; 2) transferring the parental plasmid to a host cell, inducing the parental plasmid to generate the minicircle DNA and the skeleton DNA containing the target sequence under the action of site-specific recombination; 3) subjecting the host cell to lysis, and subjecting plasmids to prepurification to obtain mixed plasmids including the minicircle DNA, the parental plasmid containing the target sequence and / or the skeleton DNA containing the target sequence; 4) removing the parental plasmid containing the target sequence and / or the skeleton DNA containing the target sequence from the mixed plasmids according to a tri-spiral purification method to obtain the high-purity minicircle DNA.

Description

technical field [0001] The invention relates to the purification of an expression vector used for gene therapy, belongs to the field of preparation of plasmid vectors, and in particular relates to a method for preparing high-purity microcircular DNA and its application. Background technique [0002] In gene therapy, an efficient and safe vector to transfer the target gene to eukaryotic cells for expression is crucial. The vectors currently used in gene therapy can be roughly divided into two categories, namely viral vectors and non-viral vectors. Among gene therapy vectors, recombinant adenoviral vectors account for 23.8%, retroviral vectors account for 20.7%, and plasmid vectors account for 18.3% (MayrhoferP et al., Methods Mol Biol, 542:87-104 (2009)). [0003] Although the transfection rate of viral vectors in vivo is high, there are some safety hazards, such as potential risks such as integration of viral genes into the host genome and immunogenicity. [0004] Compared...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/63A61K48/00C12Q1/68A61P35/00
Inventor 侯小虎何成宜陈志英
Owner SYNO MINICIRCLE BIOTECH CO LTD
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