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Mutant strain with deletion of type VI secretion systems of pseudomonas plecoglossicida and application of mutant strain

A sweetfish Pseudomonas and Pseudomonas technology, which is applied to medical preparations containing active ingredients, bacteria, antibacterial drugs, etc., can solve problems such as unsatisfactory protection effect, and eliminate the spread of a large number of virulent pathogens. possibility, improve environmental safety and controllability, and improve the effect of prevention and control

Pending Publication Date: 2020-12-11
ZHEJIANG WANLI UNIV
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  • Summary
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Studies have shown that immunizing large yellow croaker with an adjuvanted inactivated Pseudomonas ayutida vaccine can stimulate significant immunity, but the protective effect is not satisfactory; many researchers have observed that the bacteria in fish macrophages The ability to survive and proliferate in the cells suggests that the bacterium has the characteristics of a facultative intracellular pathogen. To prevent the infection of this bacterium, it is necessary to effectively stimulate the host's cellular immune response. The corresponding vaccine forms such as live attenuated vaccines and DNA vaccines may play a role better immune protection

Method used

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  • Mutant strain with deletion of type VI secretion systems of pseudomonas plecoglossicida and application of mutant strain
  • Mutant strain with deletion of type VI secretion systems of pseudomonas plecoglossicida and application of mutant strain
  • Mutant strain with deletion of type VI secretion systems of pseudomonas plecoglossicida and application of mutant strain

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Experimental program
Comparison scheme
Effect test

Embodiment 2

[0124] Embodiment 2: take large yellow croaker as the median lethal dose LD50 determination of experimental animals:

[0125] The test fish weighed 100±10g. They were temporarily raised in seawater at 20°C for 7 days, and then healthy, non-injured fish were selected and divided into 6 groups, with 10 fish in each group, and 0.2mL of the above-mentioned 104-108cells / mL bacterial suspension was injected from the chest cavity respectively. Group 6 was injected with the same dose of sterilized normal saline as a control, temporarily kept in a plastic aquarium with a diameter of 1m, inflated, changed water and absorbed dirt once a day, and was not fed. During the test, the water temperature was maintained at 20±1°C; Observe the morbidity and death of the fish, dissect the dead fish, aseptically separate and inoculate the bacteria from the spleen to the Pseudomonas color plate, confirm that the death is caused by the infection of Pseudomonas, last for 10 days, and count the deaths R...

Embodiment 3

[0129] Embodiment 3: Taking the large yellow croaker as the experimental animal's injection immune protection test

[0130] The experimental large yellow croaker was divided into 8 groups, 10 fish in each group. The prepared attenuated live vaccine was immunized by intraperitoneal injection, and the immunization dose was 10 5 -10 7 cells / tail; the control group was injected with sterile saline; two parallel groups were set up in the immune group and the control group. 4 weeks after immunization with 10 5 Cells / mL wild strain of Pseudomonas ayucidae was intraperitoneally injected to artificially attack each group of test fish, and the death and morbidity of the immune group and the control group were observed and counted within 14 days, and the immune protection rate was calculated (Table 2). The calculation formula of immune protection rate is as follows: immune protection rate%=(1-mortality rate of immunization group / mortality rate of control group)×100.

[0131] Table 2 ...

Embodiment 4

[0134] Embodiment 4: take large yellow croaker as the soaking inoculation immune protection test of test animal:

[0135] The experimental fish were randomly divided into 8 groups, 10 fish in each group. The prepared attenuated live vaccine was immunized by soaking. Dilute the prepared vaccine stock solution to 10 with sand-filtered seawater in a plastic aquarium. 5 -10 7 cells / mL concentration, soak large yellow croaker with air for 5-30 minutes, and the control group is not treated. After 4 weeks of immunization, each test group was immersed in the wild strain of Pseudomonas ayucidi (bacteria concentration 10 7 cells / mL, time 30min) for artificial infection. Observe and count the number of deaths in the control group and the immune group within 10 days, and calculate the immune protection rate (see Table 3). The calculation formula of immune protection rate is as follows: immune protection rate%=(1-mortality rate of immunization group / mortality rate of control group)×10...

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Abstract

The invention relates to a marker-free gene deletion attenuated mutant strain of a pseudomonas plecoglossicida wild virulent strain. The marker-free gene deletion attenuated mutant strain of the pseudomonas plecoglossicida wild strain can be used as an attenuated live vaccine for preventing pseudomonas plecoglossicida infection, and the marker-free gene deletion attenuated mutant strain of the pseudomonas plecoglossicida wild strain lacks partial effect element genes of three sets of type VI secretion systems of pseudomonas plecoglossicida, including T6SS1, T6SS2 and T6SS3. The pseudomonas plecoglossicida virulent strain is a pseudomonas plecoglossicida virulent strain NB2011, the preservation number of the pseudomonas plecoglossicida virulent strain NB2011 is CGMCC No.8985, the marker-free gene deletion attenuated mutant strain of the pseudomonas plecoglossicida wild virulent strain is delta T6SS1-3, and the preservation number is CGMCC No.20416. The invention also provides an application of the marker-free gene deletion attenuated mutant strain. The attenuated mutant strain provided by the invention eliminates the potential toxicity return environment and product safety risk ubiquitous in the traditional attenuated live vaccine, and is a safe, effective and economic vaccine for the visceral white spot disease of bred large yellow croaker.

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 attenuated mutant strain without a marker gene deletion of a wild strain of Pseudomonas ayucidae and related preparations and its application. Background technique [0002] Large yellow croaker (Larimichthys crocea) is an important economic fish species cultured in seawater cages in southeastern coastal provinces such as Zhejiang and Fujian. In recent years, it has been seriously harmed by the "white spot disease" of viscera. The disease usually breaks out in the low temperature season from January to March White nodules of different sizes appear in the internal organs of infected fish, such as spleen and kidney, and the affected fish basically do not eat, and cannot be effectively treated and controlled by oral medication, often resulting in extremely high ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A61K39/104A61P31/04C12R1/38
CPCC07K14/21C12N1/20A61K39/104A61P31/04A61K2039/5254A61K2039/552
Inventor 金佳敏黄梦霞李一赢毛芝娟
Owner ZHEJIANG WANLI UNIV
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