Method for simultaneously removing bacillus anthracis virulence megaplasmids pXO1 and pXO2

A Bacillus anthracis and plasmid technology, applied in the field of genetic engineering, can solve the problems of unclear replication and separation of Bacillus anthracis large plasmids, slow research progress, etc.

Inactive Publication Date: 2012-10-17
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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Problems solved by technology

However, the application of this method in Bacillus anthracis has not been reported because the replication and isolation of the large plasmid of Bacillus anthracis is not yet clear.
[0005] Efforts have been made to explain the replication and segregation of pXO1, but progress in identifying the replicon of plasmid pXO1 has been slow because this plasmid does not encode any known origin of replication proteins encoded by other plasmids. similar protein

Method used

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  • Method for simultaneously removing bacillus anthracis virulence megaplasmids pXO1 and pXO2
  • Method for simultaneously removing bacillus anthracis virulence megaplasmids pXO1 and pXO2
  • Method for simultaneously removing bacillus anthracis virulence megaplasmids pXO1 and pXO2

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Embodiment 1

[0045] Example 1. Acquisition and Identification of Recombinant Bacteria Eliminating pXO1

[0046] 1. Repel plasmid construction

[0047] 1.1 Temperature-sensitive plasmid linearization

[0048] The temperature-sensitive shuttle plasmid pKSV7 (6.9Kb) was linearized with restriction enzymes HindIII and EcoR I (HindIII and EcoR I are the multiple cloning sites of pKSV7), and then separated by 0.6% agarose gel electrophoresis, The target band was excised from the gel, and the linearized vector pKSV7 fragment was recovered with a DNA gel extraction kit, and the operation steps were performed according to the instructions.

[0049] Backbone plasmid: pKSV7 plasmid (neither transposon nor insert 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., publicly available from the Institute of Bioengineering, Academy of Military Medical Sciences, Chinese...

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Abstract

The invention discloses a method for simultaneously removing bacillus anthracis virulence megaplasmids pXO1 and pXO2. The invention provides a DNA molecule having a nucleotide sequence 1 shown in the sequence table. The invention also provides a recombinant vector, a transgenic cell line or recombinant bacteria containing the DNA molecule. The recombinant vector is prepared by inserting the DNA molecule between recognition sites HindIII and EcoRI of a shuttle plasmid. An experiment proves that according to a plasmid incompatibility principle, an incompatiple plasmid is constructed and a strain A16QT which does not contain virulence megaplasmids is obtained. The method has the characteristics of simpleness, speediness, good singularity and high safety. The method has a very important meaning for construction of a plasmid-less bacillus anthracis strain and a research on the interaction between chromosome and megaplasmids pXO1 and pXO2, provides a novel experimental means for construction of a novel vaccine, and provides a novel idea for preventing and controlling bacillus anthracis.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for simultaneously expelling the large virulence plasmids pXO1 and pXO2 of Bacillus anthracis. Background technique [0002] Bacillus anthracis is a rod-shaped, spore-forming, Gram-positive bacillus that can cause anthrax in humans and animals. If not treated in time, the mortality rate is extremely high. 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 genes. Plasmid pXO2 encodes genes involved in capsule formation and degradation. These two plasmids are crucial for 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 Bacillus anthracis has always bee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/09A61K39/02A61P31/04
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
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