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Attenuated live vaccine aiming at fish septicemia pathogen aeromonas veronii and applications thereof

A technology of Aeromonas viridis and attenuated strains, which is applied in the directions of bacteria, antitoxins, antibacterial drugs, etc., can solve the problem of less vaccines for fish, and achieve the effect of preventing and controlling infection and improving the effect of immune protection.

Inactive Publication Date: 2016-07-20
HAINAN UNIVERSITY
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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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  • Attenuated live vaccine aiming at fish septicemia pathogen aeromonas veronii and applications thereof
  • Attenuated live vaccine aiming at fish septicemia pathogen aeromonas veronii and applications thereof
  • Attenuated live vaccine aiming at fish septicemia pathogen aeromonas veronii and applications 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] SNPF1:5'-CGCGGATCCATGGGTTACCCATACGACGTTC-3'

[0027] SNPR1:5'-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 for 5...

Embodiment 2

[0103] Embodiment 2 is the pathogenic infection experiment of object with zebrafish

[0104] 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;

[0105] 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

[0106] Example 3 Determination of the half-lethal concentration LD50 of Aeromonas verkirea A.verL1 introduced into SNP-L1:

[0107] 1) Take out the SNP-L1-introduced Aeromonas veronii A. veroniiL1 stored in glycerol from -70°C, streak and activate it on the LB solid plate added with ampicillin, and culture it upside down in a 30°C incubator for 16 hours;

[0108]2) Pick a single colony of A. veroniiL1, inoculate it into LB liquid medium supplemented with ampicillin, culture it overnight at 30°C with shaking, measure the OD600, and then shake it in fresh LB culture medium with the initial OD600 of 0.0530°C until the OD600 reach 0.6-0.8, centrifuge at 5000rpm at room temperature for 15min, discard the supernatant, collect the cells, resuspend and wash with PBS once, then centrifuge at room temperature at 5000rpm for 15min, discard the supernatant, collect the cells, resuspend with PBS, and dilute tenfold. Take 10 μl of each gradient to carry out intraperitoneal injection challen...

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Abstract

The invention discloses an attenuated live vaccine aiming at fish septicemia pathogen aeromonas veronii and applications of the attenuated live vaccine. Wild-type aeromonas veronii is guided into specific pepaptamer SNP-L1 through gene modification, so that the toxicity of the wild-type aeromonas veronii is weakened, and then an aeromonas veronii attenuated strain L1 is obtained; a live vaccine prepared by the attenuated strain L1 is injected to the abdominal cavity of the zebra fish, the later-stage immune protective rate can achieve 65%, so that the occurrence of the fish bacterial septicemia can be effectively prevented, and therefore, the attenuated live vaccine has certain application values and development prospects.

Description

technical field [0001] The invention relates to a fish bacterial septicemia pathogen Aeromonas veronii (Aeromonas veronii) live attenuated 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): C12N1/21C07K14/00C12N15/11C12N15/74A61P39/02A61P31/04C12R1/01
CPCA61K39/0208A61K2039/522C07K14/00C12N15/74
Inventor 刘柱刘鹏章跃陵胡新文唐燕琼唐鸿倩
Owner HAINAN UNIVERSITY
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