Preparation method and application of Aeromonas salmonicida live vaccine preparation and freeze-dried vaccine product

A technology of Aeromonas salmonicida and live vaccines, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., which can solve the problems of restoring virulence, potential harm of exogenous gene fragment environment, and easy reversion of mutations, etc. problems, to achieve the effect of easy differentiation, possibility of eliminating virulent pathogens, and easy monitoring

Active Publication Date: 2019-03-26
马悦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the attenuated strains developed by gene recombination techniques such as using microorganisms of the same genus and different species as attenuated strains, passaged attenuated strains, pathogenic natural attenuated strains, changed in vitro cultured and passed down environments, or transposons, etc., mostly have two Congenital fatal defects: 1) Carrying resistance markers and foreign gene fragments are potentially harmful to the environment; 2) Easy to reverse mutations and restore virulence
However, there are no reports in the literature on the construction of live attenuated vaccines against Aeromonas salmonicida using these type III secretion system coding genes as deletion target genes

Method used

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  • Preparation method and application of Aeromonas salmonicida live vaccine preparation and freeze-dried vaccine product
  • Preparation method and application of Aeromonas salmonicida live vaccine preparation and freeze-dried vaccine product
  • Preparation method and application of Aeromonas salmonicida live vaccine preparation and freeze-dried vaccine product

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0036] Experimental example 1: Verification and preparation of vaccine strains

[0037] The present invention selects a wild virulent strain of Aeromonas salmonicida (a highly pathogenic strain isolated from the focus of a farmed Atlantic salmon suffering from scabies in Yantai, Shandong, my country Aeromonas salmonicida SDy0701 is the starting strain for vaccine construction, with its housekeeping gene aroC Genes and coding genes in the T3SS secretion system acr1 , aopN , ascN Is the deletion target gene, where aroC The deletion of the gene blocks the synthesis of aromatic amino acids and their folate, acr1 , aopN Is a regulatory element of the secretion system, ascN Because ATP phosphatase participates in the assembly of type III secretion system, their deletion will lead to the destruction of T3SS secretion system function and effectors. Large fragments of double deletion of key coding genes involving the colonization ability of Aeromonas salmonicida and important virul...

experiment example 2

[0059] Experimental example 2: The half-lethal dose (LD 50 ) Determination.

[0060] Breeding of experimental fish: The experimental fish (zebrafish) purchased from Jiading, Shanghai. The experimental fish were first placed in the SPF (Special Pathogen Free) laboratory to adapt to breeding for 1 week to pick out abnormal individuals. Before the infection test, the SPF test fish were stocked in a 10L infection test tank in the infection laboratory and continued to be fed for 1 week, stocking 10 fish per tank (average body length 2-3cm). The test tank uses sterile water to replace 2 / 3 of the aquaculture water every day, and the water temperature is 28.5°C with a fluctuation of 2°C.

[0061] Evaluation of virulence: randomly divide the test fish into groups, and each group will be tested in parallel with 3 tanks.

[0062] In the infection test, each group of test fish used a certain gradient dose of the virus strain: the wild strain of Aeromonas salmonicida Aeromonas salmonicida SDy07...

experiment example 3

[0071] Experimental example 3: Immunity protection test of injection administration with zebrafish as the test animal.

[0072] Evaluation of immune efficacy: test zebrafish are randomly divided into groups, each with 3 parallel tanks, 10 fish per tank. The prepared live attenuated vaccine was immunized by intraperitoneal injection. The injection dose of the immunization group is 2×10 5 CFU / tail. The control group was injected with the same volume of sterile saline. After 4 weeks of immunization, each group of zebrafish immunized with live bacteria of the wild strain of Aeromonas salmonicida (1×10 intraperitoneal injection) 4 CFU / tail) for artificial infection and challenge. Observe and count the number of deaths in the control group and the immunized group within 15 days, the statistical results are as follows image 3 Shown.

[0073] Use the following formula to calculate the relative immune protection rate:

[0074] Relative immune protection rate (RPS)% = (control group mor...

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Abstract

The invention discloses a preparation method and application of a live Aeromonas salmonicidal vaccine preparation and a freeze-dried vaccine preparation, and belongs to the technical field of fishery bacterial gene deletion live attenuated vaccines. The invention includes a markerless gene deletion attenuated mutant strain HTAsf-5307 of a wild strain of Aeromonas salmonicida, a live Aeromonas salmonicida vaccine prepared by the strain, a preparation method and application. The live attenuated vaccine strain of Aeromonas salmonicida provided by the present invention lacks some fragments of the aromatic amino acid synthesis-related genes of the Aeromonas salmonicidal virus strains, and some fragments of some type III secretion-related coding genes, relative to Its wild strain has obvious low toxicity and product safety, and at the same time, it can effectively protect immunized fish from the virus strain caused by Aeromonas salmonicida. The live vaccine has remarkable immunization effect, and can well prevent and treat diseases caused by Aeromonas salmonicida in practical application.

Description

Technical field [0001] The invention relates to a preparation method and application of a live Aeromonas salmonicida vaccine preparation and a freeze-dried vaccine product, in particular to a preparation and application based on an attenuated mutant strain of Aeromonas salmonicida without a marker gene deletion. The vaccine strain is mainly used for the immune prevention and treatment of fish scabies caused by Aeromonas salmonicida, belongs to the field of aquaculture animal disease prevention and control, and specifically belongs to the technical field of fishery bacterial gene deletion attenuated live vaccines. Background technique [0002] Aeromonas salmonicida ( Aeromonas salmonicida ) Belongs to the Aeromonas family ( Aeromonadaceae ) Aeromonas ( Aeromonas ), is a Gram-negative Brevibacterium, the optimum growth temperature is 18-22℃. Furuneulosis caused by Aeromonas salmonicida is a major disease in salmonid fish farming (Janda J.M. et al ., 2010, Clin Microbiol Rev, 23:...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21A61K39/02A61P31/04C12R1/01
CPCA61K39/02C12N1/20C12N1/205C12R2001/01
Inventor 不公告发明人
Owner 马悦
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