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Minicircle DNA and preparation method and application thereof

A microcircle and plasmid technology, applied in the field of biomedicine, can solve the problems of reliance on injection immunization, complicated purification process, and limited popularization and application, and achieve improved target gene transcription level and target protein expression, strong immune response, and good protection effect Effect

Inactive Publication Date: 2019-03-29
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although mcDNA has great advantages in the field of gene therapy and vaccines, the purification process of mcDNA is relatively complicated and depends on injection immunization, which limits its application in intensive farming.

Method used

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  • Minicircle DNA and preparation method and application thereof
  • Minicircle DNA and preparation method and application thereof
  • Minicircle DNA and preparation method and application thereof

Examples

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Embodiment 1

[0065] The present embodiment provides a method for preparing microcircle DNA, which contains a reporter gene encoding EGFP green fluorescent protein, and the preparation method comprises the following steps:

[0066] (1) Using the MN511A-1 plasmid as the basic plasmid, design primers, carry out PCR amplification, respectively introduce lox66 and lox71 sites on both sides of the CMV-EGFP-polyA reading frame, and obtain amplification product 1, wherein the primer sequences are respectively Shown in SEQ IDNO:1~4; The PCR electrophoresis figure of described amplified product one is as shown in figure 1 shown.

[0067] Wherein, the sequence of the upstream primer and the downstream primer introduced into the lox66 site are respectively shown in SEQ ID NO:1 and SEQ ID NO:2, and the sequences of the upstream primer and the downstream primer introduced into the lox71 site are respectively shown in SEQ ID NO:3 and SEQ ID NO :4,

[0068] SEQ ID NO:1

[0069] ataCCGCGGTACCGTTCGTATAAT...

Embodiment 2

[0091] The present embodiment provides the application of the microcircle DNA prepared in Example 1 and its preparation method in anti-Newcastle disease virus, and the application method is specifically:

[0092] (1) Use KpnI and NotI to double-digest pYL19 complete plasmid and pYL46 complete plasmid at the same time, after the HN gene of the target fragment gene VII type NDV is amplified by PCR with NA-HN-Kpnl-F / NA-HN-Notl-R primers , also carry out KpnI, NotI simultaneous double enzyme digestion; then the digested products of pYL19 complete plasmid and pYL46 complete plasmid were respectively connected with the digested products of HN gene amplification with T4 ligase, and pYL43 intermediate plasmids were obtained after transforming Top10 And pYL47 plasmid;.

[0093] (2) 293T cells were transfected with pYL43 plasmid and pYL47 plasmid respectively. After culture, the cells were collected to detect the expression of HN by Western-Blot, and the expression of HN protein was det...

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Abstract

The present invention provides a minicircle DNA and a preparation method and an application thereof. The minicircle DNA contains a green fluorescent protein reporter gene encoding EGFP. The preparation method comprises the following steps: lox66 and lox71 sites are respectively introduced into both sides of a CMV-EGFP-polyA reading frame; a first amplified product is subjected to SacII and StuI double enzyme digestion, and after connection, escherichia coli Top10 competent cells are transformed; pYL19 intact plasmids are subjected to PCR amplification and XmaI and PacI enzyme cutting sites areintroduced; PGK-cre-polyA plasmids are subjected to PCR amplification and XmaI and PacI enzyme cutting sites are introduced; a second amplified product and a third amplified product are subjected toXmaI and PacI double enzyme digestion, and after connection, escherichia coli Top10 competent cells are transformed; and recombinant plasmids are extracted from the pYL46 intact plasmids and then transiently transfected into well-cultured human embryonic kidney HEK-293 T cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a microcircle DNA and its preparation method and application. Background technique [0002] Minicircle DNA (mcDNA), which first appeared in 1997, is a new type of vector based on traditional plasmid DNA, which only contains the most basic eukaryotic expression framework (such as CMV promoter-target protein-polyA) and lacks prokaryotic replication Compared with plasmid DNA, it can transfer to the nucleus more efficiently and enhance transcription and translation efficiency. Compared with parental plasmids (patental plasmids, PP), mcDNA has a relatively small The expression efficiency was significantly enhanced. In addition, compared with plasmid DNA, the spatial topology of mc DNA is more stable, and more than 90% of the supercoiled structure can still be maintained in the body after immunization, while the supercoiled structure after immunization with plasmid DNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/65C12N15/66A61K48/00A61K39/17A61P31/14
CPCA61K39/12A61K48/005A61P31/14C12N15/63C12N15/65C12N15/66A61K2039/552C12N2760/18134
Inventor 姜延龙王春凤杨桂连高兴许可
Owner JILIN AGRICULTURAL UNIV
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