Nontoxic shigella flexneri with lipopolysaccharide synthesis deficiency and construction method thereof

A Shigella and Flexneri technology, applied in the field of non-toxic Shigella flexneri and its construction, can solve the problems of incomplete treatment, improper drug use of drug-resistant strains, etc.

Inactive Publication Date: 2013-01-02
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Abstract
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Problems solved by technology

At present, the main way to treat dysentery is antibiotics. Although there is no cross-immunity between different bacterial groups and serotypes, there is cross-drug resistance. In addition, the number of clinically isolated multi-drug-resistant strains continues to increase, and conventional antibiotic treatment has encountered huge challenges. ; Due to untimely treatment in the acute phase, improper use of drug-resistant strains, and incomplete treatment, etc., acute bacillary dysentery can turn into chronic bacillary dysentery, so the treatment of bacillary dysentery is increasingly challenged

Method used

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  • Nontoxic shigella flexneri with lipopolysaccharide synthesis deficiency and construction method thereof
  • Nontoxic shigella flexneri with lipopolysaccharide synthesis deficiency and construction method thereof
  • Nontoxic shigella flexneri with lipopolysaccharide synthesis deficiency and construction method thereof

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Embodiment 1

[0047] Example 1. Avirulent Shigella flexneri with lipopolysaccharide synthesis defect

[0048] 1. Preparation of avirulent Shigella flexneri 2a 301 with deletion of O-antigen ligase (waaI) gene

[0049] 1. Construction of Shigella flexneri 2a 301 virulence large plasmid deletion strain 301ΔpCP

[0050] A, Prepared Shigella flexneri 2a 301 wild strain competent

[0051]1) The wild strain of Shigella flexneri 301 (that is, Shigella flexneri 2a 301 (Shigella flexneri2a) 301, Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12and 0157, Nucl .Acids Res 2002, 30(20): 4432-4441, the public can obtain from the Institute of Bioengineering, Academy of Military Medical Sciences of the Chinese People's Liberation Army, the large virulence plasmid is pCP301) inoculated in LB liquid medium, after culturing overnight at 30°C, Transfer to 100mL low-salt LB liquid medium at a ratio of 1:100, and culture at 30°C until O...

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Abstract

The invention discloses a nontoxic shigella flexneri with lipopolysaccharide synthesis deficiency and a construction method thereof. The invention provides a recombinant bacterium A, which is a recombinant bacterium obtained by removing large virulence plasmid in shigella flexneri 2a and O-antigen ligase coding gene in genome of the shigella flexneri 2a. Experiments show that according to the method of the invention, large virulence plasmid in shigella flexneri 2a 301 is firstly removed based on a plasmid exclusion principle, and a large virulence plasmid traceless deletion mutant 301 delta pCP is constructed, which is the nontoxic shigella flexneri; a target gene is substituted by a resistance gene by using homologous recombination technology based on the action of lambda bacteriophage Red recombinase; then the resistance gene is removed from a host bacterium under the action of transposase, and thus a waaI deletion strain of the nontoxic shigella flexneri is obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a strain of avirulent Shigella flexneri deficient in lipopolysaccharide synthesis and a construction method thereof. Background technique [0002] Shigella spp., commonly known as Shigella spp., is a highly contagious Gram-negative enteropathogenic bacterium, which mainly invades human colonic epithelial cells and finally locates in the large intestine, causing typical bacillary dysentery (fever , abdominal pain, tenesmus, fecal pus and blood), and the large virulence plasmid is the most important factor of its pathogenicity, containing about 32 genes related to virulence, mainly including mxi- spa gene, ipaBCD closely related to bacterial invasion of epithelial cells, and virulence genes such as ipgC. [0003] The infection dose of Shigella is extremely low (10-100 bacteria). In the annual statutory report of infectious disease epidemics announced by the Ministry of Health of my co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/09C12N15/11C12R1/01
Inventor 王恒樑朱力宋丽冯尔玲刘先凯
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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