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Pseudomonas plecoglossicida ExoU gene knockout mutant strain and application thereof

A Pseudomonas ayumi, gene knockout technology, applied in the field of genetic engineering, can solve problems such as inability to provide effective protection

Inactive Publication Date: 2016-10-26
ZHEJIANG WANLI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the pathogenic strain of Pseudomonas ayucididae NB2011, the previous research results showed that conventional inactivated vaccines and major surface antigen immunization could stimulate a significant level of antibody response, but could not provide effective protection; Survival and proliferation in phagocytes, suggesting that the bacteria may be a facultative intracellular pathogen

Method used

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  • Pseudomonas plecoglossicida ExoU gene knockout mutant strain and application thereof
  • Pseudomonas plecoglossicida ExoU gene knockout mutant strain and application thereof
  • Pseudomonas plecoglossicida ExoU gene knockout mutant strain and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Construction of Example 1 Gene Knockout Vector

[0037] (1) According to the upstream and downstream DNA sequences of the ExoU coding gene of the P.plecoglossicida wild strain NB2011 genome, PCR-specific primers were designed, and the base sequences were as follows:

[0038] ExoU-5F:5'-ata cccggg CTGGCATTGCGGATTCCCAAG-3' (SEQ ID NO.5, the underline is the introduced SmaI restriction site)

[0039] ExoU-5R:5'-ACCCTCTCCAACATCACAATTAACTATTG-3'(SEQ ID NO.6)

[0040] ExoU-3F: 5'-TGGTTTCTTCTGATTGGCCAAGG-3' (SEQ ID NO.7)

[0041] ExoU-3R:5'-ata cccggG CTGATCGAAATGCTCTTGATCAGTC-3' (SEQ ID NO. 8, the underline is the SmaI restriction site). Use NB2011 genomic DNA as a template to amplify the target fragment by PCR. The PCR reaction system is 0.5 μL of NB2011 genomic DNA, 5 μL of 10×pfu buffer, 0.4 μL of 25 mM dNTP, ExoU-5F / ExoU-5R, ExoU-3F / ExoU-3R 0.5 μL each, 0.5 μL pfu, 43 μL double-distilled water, 50 μL in total; PCR reaction conditions: 95°C pre-denaturation for 5 mi...

Embodiment 2

[0052] Screening and Identification of Embodiment 2 Mutants

[0053] (1) Bonding test

[0054] ①Construction of donor bacteria: The targeting vector pCVD442-ΔExoU was electrotransformed into Escherichia coli β2155 strain, spread on LB plates (containing Amp 50 μg / ml, 0.5 mM DAP), and cultured at 37°C until monoclonal formation. This clone was the donor strain β2155 / pCVD442-ΔExoU used in conjugation experiments.

[0055] ② Joining test:

[0056] (1) Streak inoculate the recipient bacteria Pseudomonas ayucidida NB2011 on the LB plate, and culture at 28°C until a single colony is formed.

[0057] (2) Pick NB2011 monoclonal into 3ml LB; pick β2155 / pCVD442-ΔExoU monoclonal into 3ml LB (containing Amp25μg / ml), culture overnight at 37°C and 220rpm.

[0058] (3) Take 500 μl of donor bacteria β2155 / pCVD442-ΔExoU bacteria solution and 1000 μl of recipient bacteria (Pseudomonas ayucidida) bacteria solution and mix (volume ratio 1:2), gently pipette and mix, then centrifuge at 6000rpm ...

Embodiment 3

[0069] Embodiment 3 growth characteristics detection experiment

[0070] Under the same culture conditions, single colonies of NB2011ΔexoU (CGMCC No.) and wild strain NB2011 were picked and inoculated in 5 mL medium containing LB, and cultured overnight at 28°C with shaking. The next day, the overnight cultured bacteria were taken out, the absorbance value at 600nm was measured, and the two were diluted to about 1×10 with LB medium. 8 cells / mL concentration. Then 500 μL of the mutant strain and the wild strain were inoculated into 5 mL of LB medium, cultured at 28°C with shaking at 200 r / min, and samples were taken every 3 h for 24 h to determine the OD 600 , taking culture time as abscissa, OD 600 The value is the ordinate, and the growth curves of the mutant strain and the wild strain are drawn ( Figure 6 ), it was found that the growth rate of the mutant strain NB2011ΔexoU (CGMCC No.) was not significantly different from that of the wild strain, suggesting that ExoU had...

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Abstract

The invention belongs to the field of genetic engineering, and relates to a pseudomonas plecoglossicida ExoU gene knockout mutant strain and an application thereof. The mutant strain NB2011[delta]ExoU is preserved with preservation number of CGMCC No.12430; and a preparation method of the mutant strain comprises the following steps: conducting gene knocking-out on the ExoU gene of the pseudomonas plecoglossicida wild strain NB2011 by virtue of a homologous recombinant gene knockout technology, and conducting screening and identification by virtue of a PCR (polymerase chain reaction) technology, so that an obtained strain is determined as the gene knockout mutant strain. By researching pathogenicity of the mutant strain disclosed by the invention through animal experiments, results show that the toxicity of the mutant strain on experimental animals is significantly reduced; therefore, the mutant strain can be applicable to pseudomonas plecoglossicida hypo-toxic vaccines.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to an ExoU gene knockout mutant strain of Pseudomonas ayucididae and an application thereof. Background technique [0002] Large yellow croaker is an important economic fish species in cage culture in the east coast of my country. In recent years, it has been harmed by visceral white spot disease, resulting in high mortality and serious economic losses. Pseudomonas plecoglossicida is the pathogen of the disease. The genome sequencing and annotation results of the pathogenic strain NB2011 showed that the bacterium encoded a typical type III secretion system (type III secretion system, T3SS), which exists in many Gram-negative pathogenic bacteria, and its morphology is characterized by spanning the bacterial outer membrane A needle-shaped complex directly inserted into the eukaryotic host cell membrane, through which the bacteria transport a variety of effector proteins into the host...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/78C12N15/65A61K39/104A61P31/04C12R1/38
Inventor 毛芝娟王越峰高丽婷陈吉刚
Owner ZHEJIANG WANLI UNIV
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