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Edwardsiella tarda mutant strain and its application

A mutant strain and blunt technology, applied to Edwardsiella blunt mutant strains and their application fields, can solve the problems of the specificity and efficiency of suicide elements being difficult to control, the mutation rate of suicide loops is increased, etc., so as to improve environmental and biological safety, toxicity The effect of reducing force, protecting fish from

Active Publication Date: 2017-11-10
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned biological confinement systems all adopt complex regulatory loops, which makes it difficult to control the specificity and efficiency of the suicide elements, and at the same time increases the mutation rate of the suicide loops.

Method used

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  • Edwardsiella tarda mutant strain and its application
  • Edwardsiella tarda mutant strain and its application
  • Edwardsiella tarda mutant strain and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of markerless gene deletion attenuated mutant strain

[0044] (1) Construction of tatABCD gene deletion strain

[0045] 1) PCR amplification to obtain the desired gene fragment

[0046] Such as figure 1 As shown, with the genome of Edwardsiella tarda wild strain EIB202 (preservation number CCTCC-M 208068, the preservation place is the Chinese Type Culture Collection Center of Wuhan University, and the preservation date is May 1, 2008) as a template, using the following amplification Adding primers:

[0047] P1: GGAAGATCTCGTCTACAGCACAGCATGGA, SEQ ID NO: 1

[0048] P2: GCTTCAGCCAATCCGAACCCAGAGTACGCA, SEQ ID NO: 2

[0049] P3: GGGTTCGGATTGGCTGAAGCAGGTGACTGA, SEQ ID NO: 3

[0050] P4: ACATGCATGCATCGCTGCTGTACGCCTCTT, SEQ ID NO: 4

[0051] tatABCD-deF:CCTGTTCAATACCGCACGGCGTTTT, SEQ ID NO:5

[0052] tatABCD-deR: TGCGGCCATCCATCATATCGCTCGG, SEQ ID NO: 6

[0053] First, use P1 and P2, P3 and P4 to amplify the upstream and downstream fragments F1 and...

Embodiment 2

[0076] The preparation of embodiment 2 attenuated live vaccine

[0077] (1) Preparation of medium and saline:

[0078] 1) LB slant medium: tryptone (Difco) 10g / L, yeast extract (Merck) 5g / L, NaCl 5g / L, agar 18g / L, pH 7.5;

[0079] 2) Seed medium: tryptone (Difco) 10g / L, yeast extract (Merck) 5g / L, NaCl 5g / L, pH7.5;

[0080] 3) Fermentation medium: tryptone (Difco) 10g / L, yeast extract (Merck) 5g / L, NaCl 5g / L, pH6.8;

[0081] 4) Physiological saline: NaCl 9g / L, pH 7.2, sterilized at 121°C for 20 minutes.

[0082] (2) Preparation of vaccine composition:

[0083] Get an inoculation loop and store the attenuated vaccine strain EIBAV11092801 seeds on the LB slant medium, inoculate in a 500ml shaker flask with 100ml liquid LB seed medium, and culture with shaking at 28°C (rotating speed 200 rpm). After 12 hours, take 5ml of vigorously growing bacterial solution (OD 600 = about 4.0) were inoculated in 100 ml of fresh fermentation medium, and cultured at 28° C. for 12 hours. Was...

Embodiment 3

[0084] Example 3: Stress and virulence detection of Edwardsiella tarda strain EIBAV11092701

[0085] (1) EIBAV11092701 Osmotic stress detection

[0086] The wild strain (EIB202), the tatABCD gene deletion mutant EIBAV11092701 (ΔtatABCD) and the complementing strain (tatABCD + ) Dilute the LB culture medium cultured overnight to OD 600 =1.0, then inoculate according to 1% of the inoculum size into the LB medium containing different NaCl concentrations (1.5%, 3.0% and 3.5%, and the percentage is in g / 100ml) for cultivation, by measuring the concentration of each bacterial strain at different time points The growth status was used to detect the osmotic stress ability of EIBAV11092701 (Figure 4).

[0087] The results showed that there was no difference in growth characteristics between Edwardsiella tarda wild strain and EIBAV11092701 strain under low-salt (1.5% NaCl) conditions, while at high salt concentrations (3.0% and 3.5% NaCl), although Edwardsiella tarda wild Compared wi...

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Abstract

Provided are an Edwardsiella tarda mutant strain and application thereof, and particularly a mutant strain having a biological barrier and an attenuated mutant strain having a biological barrier.

Description

technical field [0001] The invention relates to fish vaccines and marine life safety precautions. More specifically, the present invention relates to mutants of Edwardsiella tarda with biological barriers and applications thereof. Background technique [0002] In order to realize the sustainable development of the mariculture industry and curb the increasingly serious development trend of farmed fish diseases caused by various environmental factors and the rapid increase in breeding density, the World Food and Agriculture Organization combined the successful experience of fishery development in developed countries (Ormonde P. Fisheries resources: trends in production, utilization and trade.In: Nomura I(ed.).The State of World Fishery and Agriculture 2002.Rome:FAOInformation Division, 2002, p3~p45), advocating "system management approaches" (System management approaches, SMA) The breeding mode prevents the occurrence of various diseases. One of the main contents of this mea...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N1/36A61K39/02A61P31/04A61P37/04C12R1/01
CPCA61K39/025A61K2039/522A61K2039/552A61P31/04A61P37/04C07K14/24C12N1/36
Inventor 王启要刘琴张元兴王亚敏吴海珍肖婧凡
Owner EAST CHINA UNIV OF SCI & TECH
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