Fish septicemia pathogenic aeromonas veronii attenuated live vaccine and application thereof

A technology of Aeromonas verkiserii and fish, which is applied in the field of live attenuated vaccine of Aeromonas veroris, the pathogen of fish bacterial septicemia, can solve the problem of few vaccines for fish, and achieve the prevention and control of infection, Good immune protection effect

Inactive Publication Date: 2016-09-21
HAINAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with human and veterinary vaccines, there are very few fish vaccines, especially the live attenuated vaccine of Aeromonas verkirea has not been reported at home and abroad.

Method used

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  • Fish septicemia pathogenic aeromonas veronii attenuated live vaccine and application thereof
  • Fish septicemia pathogenic aeromonas veronii attenuated live vaccine and application thereof
  • Fish septicemia pathogenic aeromonas veronii attenuated live vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation of embodiment 1 Aeromonas vernerii live attenuated vaccine

[0022] 1. Construction of pTRG-SNP-L1 recombinant plasmid

[0023] 1) PCR amplification of SNP-L1 fragment

[0024] PCR reaction system (50μl): 1×

[0025]

[0026] SNP F 1:5'-CGCGGATCCATGGGTTACCCATACGACGTTC-3'

[0027] SNP R1: 5'-CCGCTCGAGTTATCAGCGTTGTCTTCAGAC-3'

[0028] PCR reaction conditions:

[0029]

[0030] After the PCR runs, use 1% agarose gel electrophoresis to detect, and take pictures with the gel imaging system (see figure 1 ).

[0031] 2) PCR product recovery

[0032] The PCR product purification kit (spin column type) of Shanghai Jierui Bioengineering Co., Ltd. was used to purify and recover the PCR product, and eluted with 30 μl ddH2O.

[0033] 3) SNP-L1PCR purification product double digestion

[0034] Its double enzyme digestion system is as follows: 25μl

[0035]

[0036] Instantly centrifuge in a hand centrifuge for 10 sec to mix well, and digest at 37°C f...

Embodiment 2

[0100] Embodiment 2 is the pathogenic infection experiment of object with zebrafish

[0101] 1) Take out the wild-type strain Aeromonas victorii preserved in glycerol from -70°C, streak and activate it on LB solid medium supplemented with ampicillin, and culture it upside down in a 30°C incubator for 16 hours;

[0102] 2) Pick a single colony of Aeromonas victorii, inoculate it into LB liquid medium supplemented with ampicillin, culture it overnight at 30°C with shaking, measure OD600, and then in fresh LB culture medium with an initial OD600 of 0.05, 30 Shake the culture at ℃ until the OD600 reaches 0.6-0.8, centrifuge at 5000rpm at room temperature for 15min, discard the supernatant, collect the bacteria, resuspend and wash with PBS once, then centrifuge at 5000rpm at room temperature for 15min, discard the supernatant, collect the bacteria, and resuspend with PBS. Then perform ten-fold dilution, and then take 10 μl for each gradient to carry out intraperitoneal injection ch...

Embodiment 3

[0103] Example 3 Determination of the half-lethal concentration LD50 of Aeromonas verkirea A.ver L1 introduced with SNP-L1:

[0104] 1) Take out the SNP-L1-introduced Aeromonas veronii A. veronii L1 stored in glycerol from -70°C, streak and activate it on the LB solid plate supplemented with ampicillin, and culture it upside down in a 30°C incubator for 16 hours;

[0105]2) Pick a single colony of A. veronii L1, inoculate it into LB liquid medium supplemented with ampicillin, culture it with shaking overnight at 30°C, measure the OD600, and then culture it in fresh LB culture medium with an initial OD600 of 0.05 at 30°C with shaking culture When the OD600 reaches 0.6-0.8, centrifuge at 5000rpm at room temperature for 15min, discard the supernatant, collect the bacteria, resuspend and wash with PBS once, then centrifuge at 5000rpm at room temperature for 15min, discard the supernatant, collect the bacteria, resuspend with PBS, and dilute tenfold , take 10 μl for each gradient t...

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Abstract

The invention relates to a fish septicemia pathogenic aeromonas veronii attenuated live vaccine and application thereof. The fish septicemia pathogenic aeromonas veronii attenuated live vaccine has the advantages that by modifying the gene of wild aeromonas veronii and importing into a specific pepaptamer SNP-L1, the toxicity is reduced, and an aeromonas veronii attenuated strain L1 is obtained; the live vaccine prepared by the attenuated strain L1 is injected into zebra fish via abdominal cavity, the later immunity protection rate is 65percent, the fish bacterial septicemia can be effectively prevented, the certain application value is realized, and the development prospect of attenuated live vaccine is realized.

Description

technical field [0001] The invention relates to a fish bacterial septicemia pathogen Aeromonas veronii (Aeromonas veronii) attenuated live vaccine and application thereof. Background technique [0002] Since the reform and opening up, my country's aquaculture industry has developed rapidly and has made remarkable achievements. In 2014, the annual output of aquatic products was 64.5 million tons, an increase of 4.5% over the previous year. However, people are too pursuing economic benefits, constantly expanding the scale of farming, increasing the density of farming, and some unscientific management of farming models, resulting in frequent occurrence of bacterial diseases in aquaculture, which seriously limits the rapid and stable development of aquaculture. [0003] Among them, bacterial diseases caused by Aeromonas vernerii are very common in aquaculture. It is a Gram-negative rod-shaped bacterium, which is generally isolated from clinical, natural environments and food, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K39/02A61P31/04C12R1/01
CPCA61K39/0208A61K2039/522C07K14/00C12N15/74
Inventor 刘鹏刘柱章跃陵胡新文唐燕琼唐鸿倩
Owner HAINAN UNIV
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