Marker-free gene deletion attenuated mutant of vibrio alginolyticus wild strain, related preparation and application

A marker-free, alginolytic vibrio technology, applied in microorganism-based methods, medical preparations containing active ingredients, bacteria, etc., can solve the problem that the immune effect is not as good as that of live vaccines, the immune dose and cost are high, and it is not infectious. and other problems, to achieve the effect of eliminating the possibility of toxic pathogens, significant immune effect, and good control effect.

Inactive Publication Date: 2011-01-19
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, since the subunit vaccine is not infectious, it can only be immunized by injection rather than soaking, which is inconvenient to operate
And because it only has one or several antigens of the pathogen, and does not contain other antigens and genetic information of the pathogen, only a single antigen molecule is used to induce an immune response, and the immune effect is not as good as that of live vaccines
In addition, subunit vaccines need to be supplemented with adjuvants, the immunization dose and cost are relatively high, and the effect is poor

Method used

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  • Marker-free gene deletion attenuated mutant of vibrio alginolyticus wild strain, related preparation and application
  • Marker-free gene deletion attenuated mutant of vibrio alginolyticus wild strain, related preparation and application
  • Marker-free gene deletion attenuated mutant of vibrio alginolyticus wild strain, related preparation and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of markerless gene deletion attenuated mutant strain

[0031] (1) Construction of hfq gene deletion strain

[0032] 1) PCR amplification to obtain the desired gene fragment

[0033] like figure 1 As shown, using the genome of Vibrio alginolyticus strain EPGS020401 (preservation number: CCTCC No.AB209306) as a template, the following amplification primers were used:

[0034] P1(TTAGTCGACCCGACAGATGTGGGAGTATTTAGAT),

[0035] P2(TGTGGACGCTCGTCTTGTAGAGATTGCCCCTTAGCC),

[0036] P3(CTCTACAAGACGAGCGTCCACAAAGAGAAATCTGAAG),

[0037] P4(GCTACTAGTGATATCGAGAATAAGACCAGTGCGG),

[0038] First, use P1 and P2, P3 and P4 to amplify the upper and lower fragments F1 and F2 required by Overlap PCR respectively. After each fragment was recovered, the hfq deletion fragment F1F2 was obtained by using P1 and P4 using the Overlap PCR technique.

[0039] 2) Recover each fragment

[0040] To recover the target gene fragment from the object to be studied, use the glue ...

Embodiment 2

[0056] Embodiment 2: Taking zebrafish (Danio rerio) as the semi-lethal dose LD of experimental animals 50 Determination:

[0057] The fish used in the experiment were first placed in the SPF (Specific Pathogen Free) laboratory to adapt to breeding for 1 week to remove abnormal individuals. Before the infection test, the SPF test fish were stocked in a 0.5L infection test tank in the Challenge Lab, and continued to be fed for 1 week, with 10 fish (average body length 2.5-3cm, body weight 0.2g) in each tank. The test tank replaced 1 / 2 volume of culture water with sterile old water every day, and the water temperature was 28°C, with a fluctuation of 2°C.

[0058] The fish used in the test were randomly divided into groups, and two tanks were tested in parallel in each group. In the infection test, each group of test fish was treated with a certain gradient dose (10 2 -10 7 CFU / tail) of Vibrio alginolyticus wild strain and attenuated vaccine strain were artificially infected b...

Embodiment 3

[0063] Embodiment 3: Taking zebrafish as the experimental animal's immune protection test by injection

[0064] The experimental zebrafish were randomly divided into 3 groups, each with 3 parallel tanks, 10 fish per tank. The prepared attenuated live vaccine was immunized by intramuscular injection. Injection immunization dose is 7×10 5 CFU / g, intramuscular injection test zebrafish. The control group was injected with sterile normal saline. After 4 weeks of immunization, the zebrafish of each group were infected with live bacteria of the wild strain of Vibrio alginolyticus (intramuscular injection of 5×10 5 CFU / tail) for artificial infection challenge. Observe and count the control group and the number of immune deaths within 15 days to calculate the immune protection rate of each group (see Table 2).

[0065] Wherein, the immune protection rate was calculated according to the following formula: immune protection rate%=(control group death rate−immune group death rate%) / c...

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Abstract

The invention relates to a marker-free gene deletion attenuated mutant of a vibrio alginolyticus wild strain. The mutant is an attenuated live vaccine of the vibrio alginolyticus wild strain, wherein sRNA molecular chaperone protein gene hfq of the vibrio alginolyticus wild strain is deleted; and the vibrio alginolyticus wild strain is the vibrio alginolyticus wild strain EPGS020401 with a collection number of CCTCC No. AB209306. The invention also provides a preparation of the marker-free gene deletion attenuated mutant of the vibrio alginolyticus wild strain and related application to prevention and treatment of vibrio alginolyticus diseases of cultured fishes. The marker-free gene deletion attenuated mutant of the vibrio alginolyticus wild strain or the related preparation eliminates common potential security risk of environment and product existing in the conventional attenuated live vaccine, and is a safe, high-efficiency and economic vaccine aiming at the vibrio alginolyticus diseases of the cultured fishes.

Description

technical field [0001] The present invention relates to the technical field of attenuated mutant strains, in particular to the technical field of bacterial live attenuated vaccines for fish, and specifically refers to an unmarked gene-deleted attenuated mutant strain of a wild strain of Vibrio alginolyticus, related preparations and applications. Background technique [0002] With the growing population of the world, the increasing demand for seafood and the increasingly depleted natural fishery resources, the contradiction between aquaculture, as a traditional industry, has developed rapidly and effectively in modern times. Its important position has been highlighted in society, economy and people's life. According to statistics, in 2002, the total amount of aquatic products in my country accounted for 71% of the global total output and 49.8% of the total output value, ranking first in the world. According to statistics from the Ministry of Agriculture, the total output of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A61K39/106A61P31/04C12R1/63
Inventor 王启要刘欢张元兴刘琴
Owner EAST CHINA UNIV OF SCI & TECH
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