A kind of high-purity microcircle dna and its preparation method and application

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: 2018-07-27
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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  • A kind of high-purity microcircle dna and its preparation method and application
  • A kind of high-purity microcircle dna and its preparation method and application
  • A kind of high-purity microcircle dna and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0629] The construction of parental plasmids A Triplex15DsRed, Triplex15DsRed and ATriplex15DsRed, 2TT Triplex21DsRed, Triplex21DsRed with the target sequence comprises 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) , respectively adopt the following 5 pairs of primers to carry out PCR: the first pair of primers: A Triplex15-F (SEQ ID NO:41) and A Triplex-R (SEQ ID NO:44); the second pair of primers: Triplex15-F (SEQ ID NO:44); NO:21) and Triplex15-R (SEQ ID NO:22); the third pair of primers: A Triplex21-F (SEQ ID NO:42) and Triplex-R (S...

Embodiment 2

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

[0639] The plasmid ZY781.bpA p (the PspOMI and ScaI sites of MC.BESPX were inserted into the SV40bpA fragment to obtain the plasmid ZY781.bpA p, 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] 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 kan fragments with 6 Triplex15 target sequences were obtained respectively. Among them, the arrangement of the 6 Triplex15 target sequences was as follows: each of the upstream and downstream of the Kan gene had 3 tandemly repeated Triplex15 target sequences; 4 Triplex21 The kan fragment of the target sequence, wherein, the a...

Embodiment 3

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

[0646] The primer pairs CMV-F (SEQ ID NO: 25) and CMV-R (SEQ ID NO: 26), RSV-F (SEQ ID NO: 27) and RSV-R (SEQ ID NO: 27) were used, respectively, as described in Table 1. 28), Ubc-F (SEQ ID NO: 29) and Ubc-R (SEQ ID NO: 30), ApoE-F (SEQ ID NO: 31) and ApoE-R (SEQ ID NO: 32) were subjected to PCR, respectively Amplify CMV, RSV, Ubc, ApoE promoter, the genebank accession number of the template gene of described PCR amplification is respectively: 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-tripx21 through the corresponding enzyme cleavage site through conventional cloning steps. , After ide...

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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 an expression vector purified for gene therapy, belongs to the field of plasmid vector preparation, and in particular relates to a preparation method of high-purity microcircular DNA and its application. Background technique [0002] In gene therapy, an efficient and safe vector to transfer the target gene into eukaryotic cells for expression is crucial. The vectors currently used for gene therapy can be roughly divided into two categories, namely viral vectors and non-viral vectors. Among gene therapy vectors, 23.8% of recombinant adenoviral vectors are clinically used, 20.7% of retroviral vectors, and 18.3% of plasmid vectors (Mayrhofer P 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 the potential risks of viral gene integration into the host genome and immunogenicity. [0004] Compared with viral vectors, tradit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N15/63A61K48/00C12Q1/6806A61P35/00
Inventor 侯小虎何成宜陈志英
Owner SYNO MINICIRCLE BIOTECH CO LTD
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