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Avian pathogenicity escherichia coli type VI secretion system clpV gene deleted strain as well as construction method and application thereof

A technology for Escherichia coli and avian pathogenicity, which is applied in the field of avian pathogenic Escherichia coli type VI secretion system clpV gene deletion strain and its construction, and can solve problems such as no data can be found.

Pending Publication Date: 2020-07-28
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Red homologous recombination is a traditional method for gene knockout in Escherichia coli, but there is no data available for the construction of clpV gene deletion strains using it

Method used

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  • Avian pathogenicity escherichia coli type VI secretion system clpV gene deleted strain as well as construction method and application thereof
  • Avian pathogenicity escherichia coli type VI secretion system clpV gene deleted strain as well as construction method and application thereof
  • Avian pathogenicity escherichia coli type VI secretion system clpV gene deleted strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0040] 1 material

[0041] 1.1 Main instruments and equipment

[0042] MicroPulser Electroporator (Bio-Rad); PCR instrument (Bio-Rad); fluorescent quantitative PCR instrument (ABI7500).

[0043]1.2 Main strains, plasmids and reagents

[0044] Strain APEC TW-XM; plasmids pKD3, pKD46, pCP20, pBR322 (TaKaRa); sodium chloride, tryptone, yeast extract (Oxoid); ampicillin, chloramphenicol (Sangon Bioengineering Co., Ltd., Shanghai); ExTaq DNA polymerase, agarose gel DNA recovery kit, DL2000 DNA Marker, DL5000 DNA Marker (TaKaRa); L-arabinose (Sigma); Faststart Universal SYBR GREEN Master (ROX) fluorescence quantitative kit (Roche).

[0045] 1.3 Experimental animals Four-week-old ICR mice (Center for Comparative Medicine, Yangzhou University)

[0046] 2 methods

[0047] 2.1 Construction of clpV deletion strain and complementation strain

[0048] 2.1.1 Primer synthesis and design

[0049] Primers P1 / P2 were designed based on the known clpV gene sequences of APEC TW-XM strains in...

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Abstract

The invention relates to the technical field of biology, and discloses an avian pathogenicity escherichia coli type VI secretion system clpV gene deleted strain as well as a construction method and anapplication thereof, which are used for researching, preventing and controlling pathogenesis of avian pathogenicity escherichia coli. And successfully a clpV gene deleted strain of the APEC TW-XM strain is constructed by using a Red homologous recombination system to obtain the deleted strain TW-XM clpV. And the clpV gene is cloned into an expression vector pBR322, and transformed into a deletedstrain TW-XM delta clpV to obtain a corresponding gene complementary strain TW-XMC delta clpV. A mouse infected animal model is constructed by intraperitoneal injection of escherichia coli, and it isfound through detection that the bacterial load of brain, blood and lung tissue and the expression of meningitis related inflammatory factor mRNA in the brain tissue are obviously reduced. It demonstrates that clpV is closely related to APEC pathogenicity.

Description

technical field [0001] The invention relates to the field of biotechnology, and provides an avian pathogenic Escherichia coli type VI secretion system clpV gene deletion strain, a construction method and application thereof. Background technique [0002] Avian pathogenic Escherichia coli (APEC) meningitis is one of the infectious diseases harmful to the poultry industry, and there is no effective control measure so far. As a Gram-negative bacterium, Escherichia coli can release a multifunctional killing weapon T6SS (Type VI secretion system), which is widely distributed, has diverse functions, and is closely related to bacterial pathogenicity. ClpV is the ATPase of T6SS, which is crucial to the function of T6SS system. Studies have found that ClpV is an important virulence gene of Pseudomonas plecoglossicida, but whether ClpV is related to the pathogenicity of Escherichia coli and meningitis is rarely known. Research. Red homologous recombination is a traditional method fo...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70A61K39/108A61P31/04C12R1/19
CPCC07K14/245C12N15/70A61K39/0258A61P31/04A61K2039/522
Inventor 孟霞王亨钟昊然李建基朱国强陈艳飞
Owner YANGZHOU UNIV
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