Application of recombinant vibrio parahaemolyticus flagellin in improvement of fish immunity

A technology of Vibrio hemolyticus and flagellin is applied in the field of genetic engineering to achieve the effects of improving immunity, improving fish immunity and good application value

Pending Publication Date: 2020-12-01
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing methods for the prevention and control of Vibrio parahaemolyticus infection in fish, and to provide the application of recombinant Vibrio parahaemolyticus flagellin in improving the immunity of fish

Method used

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  • Application of recombinant vibrio parahaemolyticus flagellin in improvement of fish immunity
  • Application of recombinant vibrio parahaemolyticus flagellin in improvement of fish immunity
  • Application of recombinant vibrio parahaemolyticus flagellin in improvement of fish immunity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035]Cloning of embodiment 1 Vibrio parahaemolyticus flagellin FlaA, FlaB, FlaC, FlaD, FlaE, FlaF genes

[0036] 1. Experimental method

[0037] (1) Extraction of Vibrio parahaemolyticus DNA

[0038] We used the Bacterial Genomic DNA Extraction Kit (Tiangen, China) to extract the total DNA of Vibrio parahaemolyticus, and the operation steps were as follows:

[0039] ① Take 3 mL of Vibrio parahaemolyticus, centrifuge at room temperature at 10,000 rpm for 1 min, suck off the supernatant as much as possible, add 200 μL buffer GA to the cells, and shake until the cells are completely suspended.

[0040] ②Add 20 μL proteinase K to the tube and mix well.

[0041] ③ Add 220 μL buffer GB, shake for 15 seconds, place at 70°C for 10 minutes until the solution becomes clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0042] ④ Add 220 μL of absolute ethanol, vortex and mix well for 15 seconds, when a flocculent precipitate appears, centrifuge ...

Embodiment 2

[0057] Embodiment 2 Construction of recombinant Vibrio parahaemolyticus flagellin FlaA, FlaB, FlaC, FlaD, FlaE, FlaF prokaryotic expression vector

[0058] 1. Experimental method

[0059] (1) In order to introduce the His fusion tag, first clone the ORF fragments encoding the Vibrio parahaemolyticus flagellin FlaA, FlaB, FlaC, FlaD, FlaE, and FlaF into the pET28a(+) vector (the map of the pET28a vector is as follows figure 2 Shown), the prokaryotic expression vectors pET28a-flaA, pET28a-flaB, pET28a-flaC, pET28a-flaD, pET28a-flaE, pET28a-flaF of His fusion tag were constructed. The primers and their sequences used to construct the above prokaryotic expression vectors are shown in Table 2.

[0060] (2) The complete ORF coding sequence with the fusion tag His was amplified with the primers shown in Table 2, and cloned into pET28a(+) vector by CloneExpressII One Step Cloning Kit (Novazyme, China).

[0061] Table 2

[0062]

[0063] 2. Experimental results

[0064] The pro...

Embodiment 3

[0065] Prokaryotic expression of embodiment 3 recombinant Vibrio parahaemolyticus flagellin FlaA, FlaB, FlaC, FlaD, FlaE, FlaF

[0066] 1. Experimental method

[0067] (1) The correct prokaryotic expression vector pET28a-flaA obtained in Example 2, pET28a-flaB, pET28a-flaC, pET28a-flaD, pET28a-flaE, pET28a-flaF are transformed into Escherichia coli BL21 (DE3), through LB Kan﹢ Plates were incubated overnight at 37°C.

[0068] (2) Pick a single colony in 5mL LB Kan﹢ In liquid medium, activate overnight at 37°C, 200rpm.

[0069] (3) Inoculate 50 mL of fresh LB at a ratio of 1:100 Kan﹢ In liquid medium, culture at 37° C. and 200 rpm for 2.5 hours so that OD600=0.6-0.7.

[0070] (4) Add IPTG to a final concentration of 0.5 mM, induce culture at 25° C. and 200 rpm for 4 h.

[0071] (5) Take 500 μL of bacterial liquid, collect the bacterial cells by centrifugation at 4°C, 10,000 rpm, resuspend the bacterial cells with 40 μL of PBS, add 5×SDS loading buffer, treat at 100°C for 1...

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Abstract

The invention discloses application of recombinant vibrio parahaemolyticus flagellin in improvement of fish immunity. It is found that the recombinant vibrio parahaemolyticus flagellin can induce expression of related immune genes and participate in activation of corresponding immune signal channels, the immunity of fishes is remarkably improved, the recombinant vibrio parahaemolyticus flagellin is expected to be developed into a natural fish immunopotentiator or immunologic adjuvant, and then fish diseases caused by vibrio parahaemolyticus are prevented or treated. Therefore, the recombinantvibrio parahaemolyticus flagellin has a good application prospect in improving fish immunity and preparing a fish immunopotentiator or immunologic adjuvant.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. More specifically, it relates to the application of recombinant Vibrio parahaemolyticus flagellin in improving fish immunity. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a Gram-negative, halophilic, mesophilic, rod-shaped bacterium commonly found in tropical and temperate oceans and naturally distributed in coastal habitats and estuarine areas around the world. Often isolated from a variety of seafood including cod, sardines, mackerel, flounder, clams, octopus, shrimp, crab, lobster, scallops and oysters. This bacterium is thought to be a major cause of gastroenteritis, wound infection and sepsis in humans. Consumption of raw or undercooked seafood, especially shellfish contaminated with Vibrio parahaemolyticus, may lead to the development of acute gastroenteritis characterized by diarrhea, headache, vomiting, nausea, abdominal cramps, and low-...

Claims

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Application Information

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IPC IPC(8): A61K38/16A61K39/39A61K39/106A61P37/04A61P31/04
CPCA61K38/164A61K39/39A61K39/107A61P37/04A61P31/04
Inventor 卢丹琪于雪何良格张勇林浩然
Owner SUN YAT SEN UNIV
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