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Construction method of aeromonas veronii attenuated strain, strain and application thereof

A technology for the construction of Aeromonas verdeii, which is applied in the field of construction of attenuated strains of Aeromonas verdeii, which can solve problems such as drug residues and environmental pollution, and achieve the effect of reduced toxicity and good attenuation effect

Active Publication Date: 2020-03-06
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, we mainly rely on antimicrobial drugs to prevent and control Aeromonas victorii, but drug control methods directly lead to the formation of drug-resistant pathogens, and at the same time cause many environmental problems such as drug residues and environmental pollution.

Method used

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  • Construction method of aeromonas veronii attenuated strain, strain and application thereof
  • Construction method of aeromonas veronii attenuated strain, strain and application thereof
  • Construction method of aeromonas veronii attenuated strain, strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of Aeromonas vernerii exsA knockout strain using homologous double exchange technology

[0052] 1. Design primers and amplify homology arm fragments

[0053] Without affecting the exsA upstream gene and downstream gene coding, this application selects the sense strand of the DNA sequence 2009697-2010213 including the exsA start codon, with a total of 516 bp nucleotides as the knockout fragment. Design primers exsA-F for its upstream homology arm and downstream homology arm respectively 1 / R 1 and exsA-F 2 / R 2 , and a restriction endonuclease BstXI restriction site was added to the primer, and the recognition sequences of the restriction endonucleases KpnI and SacI required for double digestion of the vector were added to the middle sequence of the BstXI enzyme recognition site. Wherein, the DNA sequence of the upstream homology arm is shown in SEQ ID NO.4, and the DNA sequence of the downstream homology arm is shown in SEQ ID NO.5. The desi...

Embodiment 2

[0069] Example 2: Virulence detection of Aeromonas verkisii exsA knockout strain

[0070] 1. Growth curve determination

[0071] image 3 The growth curves of C4 wild type and exsA knockout type of Aeromonas verkisei under the same conditions. As shown in the figure, compared with C4 wild type, C4-ΔexsA had no significant difference in the growth rate, reproductive ability, perception and absorption of external nutrients. It indicated that the exsA gene had no obvious effect on the growth of Aeromonas verkistan.

[0072] 2. Detection of C4-ΔexsA on tilapia half-lethal concentration (LD50)

[0073] The virus challenge experiment was carried out on tilapia. In the experiment of this application, the method of intraperitoneal injection of tilapia was used to challenge the virus. Divide tilapia into experimental group and control group, with at least 80 fish in each group. The tilapia of the experimental group were injected with C4-ΔexsA of Aeromonas verkisii, while the tila...

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Abstract

The invention relates to aeromonas veronii, which is preserved in China Center for Type Culture Collection (CCTCC) on July 24th, 2019, and a preservation number of the aeromonas veronii is CCTCC NO: M2019580. The virulence of the aeromonas veronii mutant strain is greatly reduced, a good attenuating effect is achieved, and possibility is provided for preparing aeromonas veronii attenuated live vaccines.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for constructing an attenuated strain of Aeromonas vernerii, a strain and an application thereof. Background technique [0002] Aeromonas victorii belongs to the Aeromonas family, the genus Aeromonas, and is a Gram-negative short bacillus. It is ubiquitous in fresh water, sewage, sea water and even soil. Aeromonas verdeii is a virulent pathogenic bacteria. The pathogenic process mainly includes adhesion-invasion-colonization in the host body and toxin secretion, during which a series of virulence factors are produced, such as aerolysin, enterotoxin, etc. ) and adhesion factors, etc. These virulence factors play a pivotal role in aquatic animals, livestock and poultry, and even human infection. [0003] In recent years, an increasing number of cases have shown that Aeromonas victorii has become an important human-fish pathogenic bacteria, which is widely distributed, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K39/02A61P31/04C12R1/01
CPCA61K39/02A61K2039/522A61P31/04C07K14/195
Inventor 李宏徐一轲刘柱马香唐燕琼盛强龙孙愉宸
Owner HAINAN UNIVERSITY
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