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Method for removing pxo2 plasmid in bacillus anthracis

A Bacillus anthracis and plasmid technology, applied in the biological field, can solve the problems of poor specificity, possible removal of other plasmids other than the target plasmid, random mutation of host cells, etc., to achieve the effect of simple operation

Active Publication Date: 2021-07-09
ACADEMY OF MILITARY MEDICAL SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, these methods have potential problems. The first is poor specificity, that is, other plasmids other than the target plasmid may be removed during the process of removing the target plasmid, and the second is the possibility of random mutations in the host cell during the treatment process.

Method used

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  • Method for removing pxo2 plasmid in bacillus anthracis
  • Method for removing pxo2 plasmid in bacillus anthracis
  • Method for removing pxo2 plasmid in bacillus anthracis

Examples

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Embodiment 1

[0076] Example 1. Using the CRISPR / Cas9 system to remove the Bacillus anthracis pXO1 plasmid and pXO2 plasmid

[0077] 1. Construction of plasmids ("scissor plasmids") containing the CRISPR / Cas9 system and target sequences

[0078] 1.1 Sequence design of pXO1 plasmid and pXO2 plasmid target sequence (N20)

[0079] Use a sequence (ORF16) on the Bacillus anthracis virulence large plasmid pXO1 (GenBank accession no.AF065404) as the target sequence; use a sequence (GBAA_pXO2_0038) on the Bacillus anthracis virulence large plasmid pXO2 (GenBank accession NO.NC_007323) For the target sequence, use the software sgRNAcas9_V3.0_GUI (or other software) to design the N20-specific target sequence in the sgRNA, mark the target sequence on pXO1 as O1T (the 1st-20th position of sequence 3 in the sequence table), and put the target sequence on pXO2 The target sequence is marked as O2T (No. 1-20 of Sequence 4 in the Sequence Listing), and its sequence is shown in Table 1. The two target sequ...

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Abstract

The invention discloses a method for removing pXO2 plasmid in bacillus anthracis. The method for removing the pXO2 plasmid in Bacillus anthracis disclosed in the present invention is completed by using the CRISPR / Cas9 system. The CRISPR / Cas9 system contains an sgRNA named sgRNA2, and the target sequence recognized by sgRNA2 is a specific gene contained in the pXO2 plasmid but not contained in Bacillus anthracis. Sequence, the target sequence is the DNA fragment shown in the 1-20th position of sequence 4 in the sequence listing. The present invention uses the CRISPR / Cas9 system to remove the pXO2 plasmid, which is a large virulence plasmid of Bacillus anthracis, and successfully obtains Bacillus anthracis that does not contain the pXO2 plasmid. More convenient and new means provide a new idea for the prevention and treatment of Bacillus anthracis.

Description

technical field [0001] The invention relates to a method for removing pXO2 plasmid in bacillus anthracis in the field of biotechnology. Background technique [0002] Bacillus anthracis (also known as Bacillus anthracis) is a Gram-positive aerobic bacillus that can form spores, which can cause anthrax in humans and animals. If it is not treated in time, the mortality rate is extremely high, causing great economic losses. life-threatening. Bacillus anthracis contains two large virulence-related plasmids: pXO1 (181.6kb) and pXO2 (96.2kb). Plasmid pXO1 encodes protective antigen, lethal factor and edema factor and other anthrax toxin proteins and their regulatory proteins. Plasmid pXO2 encodes genes involved in capsule formation and degradation. These two plasmids are crucial to the pathogenicity of Bacillus anthracis, and the loss of any one of the plasmids will greatly reduce the virulence of Bacillus anthracis. Therefore, the research on the large virulence plasmid of Bac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/75C12N15/113C12N1/21C12R1/07
Inventor 刘先凯王东澍冯尔玲王晓景吕宇飞潘超朱力王恒樑
Owner ACADEMY OF MILITARY MEDICAL SCI
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