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Method for eliminating multi-copy plasmid in salmonella by using suicide vector

A suicide vector, Salmonella technology, applied in the field of microorganisms, can solve problems such as differences in effectiveness and gene mutation of host bacteria, and achieve the effect of simple process

Active Publication Date: 2017-07-21
YANGZHOU UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The current plasmid elimination methods mainly use chemical elimination agents (chimeric dyes, coumarin, novobiocin, rifampicin, mitomycin C, sodium dodecyl sulfate) and physical elimination methods (high temperature, high Piezoelectric poration, protoplasts), and molecular biology elimination methods (transposons, plasmid incompatibility) to achieve the elimination of plasmids, but these methods are only effective for some plasmids in some strains, that is, in different strains There is great variation in the effectiveness of these plasmid depletion methods between , or between different plasmids
In addition, many eliminators themselves are also mutagens, which can easily cause mutations in the genes of host bacteria.

Method used

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  • Method for eliminating multi-copy plasmid in salmonella by using suicide vector
  • Method for eliminating multi-copy plasmid in salmonella by using suicide vector
  • Method for eliminating multi-copy plasmid in salmonella by using suicide vector

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Construction of pSPI12 plasmid elimination mutant strain of embodiment 1 S06004

[0045] Salmonella Pullorum S06004 Source: Zoonosis Laboratory of Yangzhou University.

[0046] 1. Identification of S06004:

[0047] 1.1. Bacteria identification

[0048] 1.1.1 Biochemical test

[0049] Pick a purely cultured single colony and inoculate it into a biochemical identification tube, and observe the results after incubating at 37°C for 24 hours.

[0050] 2. Construction of pSPI12 plasmid elimination mutant strain of S06004

[0051] 2.1 Target gene sequence determination

[0052] 2.1.1 Amplification of target genes

[0053] According to the Salmonella genome sequence published on GenBank, specific primers were designed with the primer design software Primer5.0, as shown in Table 2. Genomic DNA of Salmonella pullorum S06004 was used as a template to amplify the upstream gene fragment of ipaJ by PCR method.

[0054] The 50μl PCR amplification system is as follows:

[0055]...

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Abstract

The invention belongs to the field of microorganisms, and in particular relates to a method for eliminating multi-copy plasmid in salmonella by using a suicide vector, and application of the method. According to the method, the multi-copy plasmid in the salmonella is eliminated by using an exogenous suicide vector. The whole process is simple, rapid, stable and efficient. A basis is made for studying functions of the plasmid.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a method for eliminating multi-copy plasmids in Salmonella by using suicide vectors and an application thereof. Background technique [0002] Salmonella is an important zoonotic pathogen and one of the most important foodborne pathogens in the world, which has important public health significance. At the same time, Salmonella is an important pathogen of livestock and poultry, which is extremely harmful to the livestock and poultry breeding industry. At present, new effective control measures need to be established. Therefore, it is of great significance to study its pathogenic mechanism for the effective prevention and treatment of Salmonella infection. [0003] Plasmid is the genetic material independent of extrachromosomal in bacterial cells, often exists in the cytoplasm, is a double-helix, closed-circle DNA molecule, which can replicate itself and be passed on from ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12R1/42
CPCC12N9/1055C12N15/74C12Y204/0101
Inventor 焦新安李求春尹超徐黎娟尹克全胡亚辰孙林李扬陈祥潘志明耿士忠
Owner YANGZHOU UNIV
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