US 2026/0226498 A1 · Antibiotic-free Miniplasmid, and Preparation Method Therefor and Use Thereof

Abstract

The present application provides an antibiotic resistance gene-free miniplasmid, comprising a nucleotide sequence encoding an antitoxin protein, and a replicon. A backbone of the antibiotic resistance gene-free miniplasmid of the present application is smaller and controlled within 1,000 bp, redundant useless fragments are reduced, and meanwhile, the miniplasmid does not comprise a resistance expression cassette, thereby improving the utilization rate of a sequence of interest in the plasmid and reducing the production burden. The miniplasmid has higher efficiency and safety in the plasmid DNA-mediated non-viral vector delivery, gene therapy, vaccine immunity, virus production, antibody production, and other genetic engineering. The present application further provides a system for producing a plasmid based on a toxin-antitoxin system, a bacterial host cell comprises a gene expression cassette for inducibly expressing a toxin protein, the antibiotic resistance gene-free miniplasmid comprises a gene expression cassette for expressing an antitoxin protein, and the system is used for efficiently maintaining replication and amplification of the antibiotic resistance gene-free miniplasmid.

Claims29

Dependent claims

An antibiotic resistance gene-free miniplasmid, comprising a nucleotide sequence encoding an antitoxin protein, and a replicon, wherein

an amino acid sequence of the antitoxin protein comprises the following sequence:
(1) an amino acid sequence shown in SEQ ID NO:
1, or an amino acid sequence having one or more mutations of E24D, I36V and V43I compared with SEQ ID NO:
1;
or (2) an amino acid sequence shown in SEQ ID NO:
9, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A and N54K compared with SEQ ID NO:
9;
and
a length of the replicon is ≤800 bp, preferably ≤600 bp or ≤300 bp.

6 more elements

A system for producing a plasmid, comprising a host cell and a plasmid, wherein the host cell comprises a nucleotide sequence encoding a toxin protein, and the plasmid comprises a nucleotide sequence encoding an antitoxin protein;

an amino acid sequence of the toxin protein is shown in SEQ ID NO:
4, and an amino acid sequence of the antitoxin protein comprises:
an amino acid sequence shown in SEQ ID NO:
1, or an amino acid sequence having one or more mutations of E24D, I36V and V43I compared with SEQ ID NO:
1;
or
an amino acid sequence of the toxin protein is shown in SEQ ID NO:
13, and an amino acid sequence of the antitoxin protein comprises:
an amino acid sequence shown in SEQ ID NO:
9, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A and N54K compared with SEQ ID NO:
9.

8 more elements

Dependencies are inferred from the claim text.

Antibiotic-free Miniplasmid, and Preparation Method Therefor and Use Thereof

Life of this patent

Antibiotic-free Miniplasmid, and Preparation Method Therefor and Use Thereof

Life of this patent

Abstract

Claims29