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A method for expelling a bacillus anthracis virulence large plasmid pXO1

A technology of bacillus anthracis and plasmid, which is applied in the field of genetic engineering, can solve the problems of unavailability of strains, random mutation of host bacteria chromosomes, etc., and achieves the effects of high safety and good specificity

Inactive Publication Date: 2014-04-30
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In these methods of expelling pXO1, the loss of pXO1 is random and must go through a large number of screening processes, and sometimes even the desired strain cannot be obtained. In addition, the random mutation of the host bacterial chromosome may be caused during the screening process

Method used

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  • A method for expelling a bacillus anthracis virulence large plasmid pXO1
  • A method for expelling a bacillus anthracis virulence large plasmid pXO1
  • A method for expelling a bacillus anthracis virulence large plasmid pXO1

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Experimental program
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Embodiment 1

[0039] Example 1. Obtainment and identification of recombinant bacteria that expelled pXO1

[0040] 1. Repel plasmid construction

[0041] 1.1 Linearization of temperature-sensitive plasmids

[0042] The temperature-sensitive shuttle plasmid pKSV7 (6.9Kb) was linearized with restriction enzyme SmaI, and then separated by 0.6% agarose gel electrophoresis, the target band was cut from the gel, and the DNA gel recovery kit was used The linearized vector pKSV7 fragment was recovered, and the operation steps were performed according to the instructions.

[0043] Skeleton plasmid: pKSV7 plasmid (no transposon nor inserted sequence on it): Smith K, Youngman P. Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene[J]. Biochimie, 1992 , 74: 705-711. The public can obtain it from the Institute of Bioengineering of the Chinese Academy of Military Medical Sciences.

[0044] 1.2. PCR to get rid of fragments

[0045] Using the genome of...

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Abstract

The invention discloses a method for expelling a bacillus anthracis virulence large plasmid pXO1. A recombinant vector provided in the invention is a vector acquired by inserting DNA molecules into a Sma1 locus of a shuttle plasmid pKSV7, and a nucleotide sequence of the DNA molecules is a sequence 1 of a sequence table. Experiments in the invention prove that the method constructs an incompatible plasmid based on a plasmid exclusion principle, that the incompatible plasmid specifically expels the virulence large plasmid pXO1 from a bacillus anthracis A16R (pXO1+pXO2-), that the obtained product is cultivated at 37 DEG C and introduced exogenous plasmids are expelled, and that a bacterial strain A16RO is obtained to expel the pXO1. The method has characteristics of simplicity, fast speed, good specificity, and high safety. The method is of great significance for construction of bacillus anthracis plasmid-lacking stains and a research on interactions between the large plasmid pXO1 and chromosomes. The method provides new experimental means for constructing new vaccine strains and new ideas for preventing and curing the bacillus anthracis.

Description

Technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for repelling the virulent plasmid pXO1 of Bacillus anthracis. Background technique [0002] Bacillus anthracis is a rod-shaped, spore-forming Gram-positive bacillus that can cause anthracnose in humans and animals. If it is not treated in time, the mortality rate is extremely high. Bacillus anthracis contains two virulent plasmids related to pathogenicity: pXO1 (181.6kb) and pXO2 (96.2kb). Plasmid pXO1 encodes anthrax toxin proteins such as protective antigens, lethal factors and edema factors and their regulatory genes. The plasmid pXO2 encodes genes involved in capsule formation and degradation. These two plasmids are critical to the pathogenicity of Bacillus anthracis, and the loss of any plasmid will cause the virulence of Bacillus anthracis to be greatly reduced. Therefore, the research on the virulent plasmid of Bacillus anthracis has always been a research hotspo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/09C12N1/21A61K39/02A61P31/04
Inventor 王恒樑刘先凯王东澍王华贵冯尔玲
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE