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Application and construction method of Vulpine Escherichia coli htra (high temperature requirement A) gene deleted strain

A technology of Escherichia coli and gene deletion, applied in application, genetic engineering, plant genetic improvement, etc., to achieve the effect of short experimental period, decreased anti-phagocytic phagocytic ability, and high cell efficiency

Inactive Publication Date: 2018-08-03
HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, for pathogenic Escherichia coli, the systematic study of the relationship between HtrA protein and virulence has not been reported

Method used

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  • Application and construction method of Vulpine Escherichia coli htra (high temperature requirement A) gene deleted strain
  • Application and construction method of Vulpine Escherichia coli htra (high temperature requirement A) gene deleted strain
  • Application and construction method of Vulpine Escherichia coli htra (high temperature requirement A) gene deleted strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Design of λ-Red Homologous Recombination Primers

[0056] Fox Escherichia coli isolate HBCLE-12(WT) was isolated from a sick silver fox in a fur animal farm in Changli. The sick fox showed acute pneumonia and sepsis. Staining, identification of biochemical characteristics, and 16S-rDNA sequence analysis identified Escherichia coli, and its serotype was determined to be O1. The isolate was preserved by the Laboratory of Animal Infectious Diseases, Hebei Science and Technology Normal University.

[0057] The htra gene isolated from the fox-derived Escherichia coli HBCLE-12 strain was sequenced and analyzed, and primers were designed, wherein the homologous sequence of the htra gene was used as a template, and the homologous sequence of the htra gene was SEQ ID NO:1. The underlined part at the 5' end of primers P1 and P2 is homologous to the gene sequence to be knocked out, and the ununderlined part at the 3' end is complementary to the sequences on both sides of the chlor...

Embodiment 2

[0063] Construction of htra gene-deleted strain of Escherichia coli from fox

[0064] Using the calcium chloride method, the plasmid pKD46 was transferred into the fox-derived Escherichia coli isolate HBCLE-12, the ampicillin-resistant bacterial strain was picked, identified by primers P3 and P4, and the positive bacterial strain was inoculated in the culture medium containing ampicillin. In LB medium, the concentration of ampicillin is 30ug / mL. Cultivate at 30°C, and add L-arabinose at a final concentration of 30mmol / L to induce the culture, until the OD of the bacterial solution 600 Competent cells were prepared when the value reached 0.4-0.6;

[0065] Purify the gene fragments containing htra gene homologous sequences amplified by P1 and P2, and introduce the purified gene fragments containing htra gene homologous sequences into the competent cell one by electrotransformation, wherein The electroporation conditions are voltage 1.8KV, pulse 25μF, resistance 200Ω; pick chlo...

Embodiment 3

[0067] Construction of complementing strains

[0068] Extract the genome of the fox-derived Escherichia coli HBCLE-12, use the genome of the fox-derived Escherichia coli HBCLE-12 as a template, use primers P5 and P6 to amplify the complete open reading frame of the htra gene, and clone it into the plasmid pBR322; The complementing plasmid pBR322-htra was electrotransformed into the HBCLE-12Δhtra mutant strain to construct the complementing strain. The present invention utilizes Western blot to detect the effect of replenishing bacterial strains, and the results are as follows: figure 2 , wherein the primary antibody is a His-tagged Escherichia coli HtrA protein polyclonal antibody at a concentration of 1:1000; the secondary antibody is an HRP-labeled goat anti-mouse IgG antibody at a concentration of 1:5000. Depend on figure 2 It can be seen that the complementing strain can express HtrA protein.

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Abstract

The invention discloses an application and a construction method of a vulpine Escherichia coli htra (high temperature requirement A) gene deleted strain. Virulence of Escherichia coli can be reduced by vulpine Escherichia coli htra gene deletion, and the vulpine Escherichia coli htra gene deleted strain can be applied to an Escherichia coli vaccine. The vulpine pneumonia Escherichia coli htra genedeleted strain is constructed with a Red homologous recombination method, and the mutant strain is evaluated from growth characteristics, biochemical characteristics, capability of resisting phagocytosis of phagocytes, virulence and other aspects. The vulpine Escherichia coli htragene deleted strain is constructed successfully, which proves that htra protein can affect virulence of Escherichia coli, the foundation is laid for researching action mechanism of the htra protein, and reference is further provided for designing an Escherichia coli vaccine target.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to the application and construction method of a fox-derived Escherichia coli htra gene deletion strain. Background technique [0002] In recent years, with the increasing scale and intensification of fur animal breeding and the increasing stocking density, acute pneumonia caused by various pathogenic bacteria has increased sharply, causing a large number of fur animals to die and bringing heavy losses to the fur industry. The epidemiological survey of bacterial pneumonia in foxes and raccoons showed that pneumonia caused by Escherichia coli infection accounted for a considerable proportion, and there was an upward trend in recent years. Escherichia coli can cause pneumonia in foxes, raccoons and minks, mostly in summer and autumn, especially in the moulting season, and young animals are susceptible, mainly causing sepsis, respiratory and nervous system damage, and adult ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/31A61K39/02A61P31/04
CPCA61K39/0258A61P31/04C12N15/70C07K14/245A61K2039/552
Inventor 张志强史秋梅吴同垒高桂生杨楠李巧玲
Owner HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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