1. An attenuated strain of Edwardsiella, wherein a target gene or protein encoded thereby is down-regulated, said target gene comprising: eseg, EseJ, EseH, ETAE _1586, ETAE _1604, ETAE _2186, ETAE _2188, ETAE _3282, EvpJ.

2. The attenuated strain of Edwardsiella according to claim 1, wherein said target gene further comprises: rpoS; and/or

Expression of the esrB gene is also up-regulated in the attenuated strain of Edwardsiella.

3. The attenuated strain of Edwardsiella according to any of claims 1 to 2, wherein said down-regulation comprises: knocking out or silencing a target gene, or inhibiting the activity of a protein encoded by a target gene; preferably, it comprises: knocking out a target gene by a homologous recombination method, knocking out the target gene by a gene editing method, silencing the target gene by a site-specific mutation method, or silencing the target gene by an interference molecule expressed by a specific interference gene; preferably, the homologous recombination method comprises a marker-free in-frame deletion mutation method; or

Driving expression of the esrB gene with a strong promoter; preferably, the strong promoter comprises p_rpsUA promoter.

4. The attenuated strain of Edwardsiella according to claim 1, wherein said Edwardsiella comprises: edwardsiella pisciida, Edwardsiella tarda, Edwardsiella ictaluri; preferably, the Edwardsiella piscicida comprises strain EIB 202.

5. Use of the attenuated strain of edwardsiella according to any of claims 1 to 4 for the preparation of a composition for inhibiting edwardsiella; preferably the composition is a vaccine composition; or

Used for inducing the Edwardsiella host to generate immune protection against the Edwardsiella.

6. The use according to claim 5, wherein the attenuated strain induces immunoprotection against Edwardsiella in fish; preferably, the fish is fish with Edwardsiella as a host; more preferably, the fish comprises: fish of the order flounders, carpiformes, perciformes; more preferably, the method comprises the following steps: fish of Paralichthys of Pleuronectiformes such as Scophthalmus maximus, and fish of Cyprinidae such as zebrafish, catfish, eel, Paralichthys, salmon, and tilapia.

7. A composition comprising the attenuated strain of Edwardsiella according to any of claims 1 to 4 and a pharmaceutically acceptable carrier; preferably, the composition is a vaccine composition.

8. A method of reducing the toxicity or making an attenuated strain of edwardsiella, comprising: down-regulating a target gene or a protein coded by the target gene in a genome of the plant, wherein the target gene comprises: EseG, EseJ, EseH, ETAE _1586, ETAE _1604, ETAE _2186, ETAE _2188, ETAE _3282, EvpJ; preferably, the target gene further comprises: rpoS; more preferably, the expression of the esrB gene is also up-regulated in said attenuated strain of Edwardsiella.

9. The method of claim 8, wherein said down-regulating comprises: knocking out or silencing a target gene, or inhibiting the activity of a protein encoded by a target gene; preferably, it comprises: knocking out a target gene by a homologous recombination method, knocking out the target gene by a gene editing method, silencing the target gene by a site-specific mutation method, or silencing the target gene by an interference molecule expressed by a specific interference gene; preferably, the homologous recombination method comprises a marker-free in-frame deletion mutation method; more preferably:

1-6 primers of SEQ ID NO are used for targeting deletion of ETAE _1586,

43-54 of the primers of SEQ ID NO are used for targeting deletion of EseJ,

primers of SEQ ID NO 13-18 are used for targeting deletion of EseH,

primers of SEQ ID NO 55-60 are used for targeting deletion, EseG,

primers of SEQ ID NO 7-12 are used for targeting deletion of ETAE _1604,

the primers of SEQ ID NO 19-24 are used for targeting deletion of ETAE _2186,

25-30 of primers of SEQ ID NO are used for targeting deletion of ETAE _2188,

the primers of SEQ ID NO 37-42 are used for targeting deletion of ETAE _3282,

primers of SEQ ID NO. 31-36 are used for targeting deletion of EvpJ,

primers of SEQ ID NO 61-66 are used for targeted deletion of rpoS.

10. A method of increasing immunoprotection in fish, comprising: administering to a fish an attenuated strain of edwardsiella according to any one of claims 1 to 4, or a composition according to claim 8; preferably, the fish is fish with Edwardsiella as a host; more preferably, the fish comprises: fish of the order flounders, carpiformes, perciformes; more preferably, the method comprises the following steps: fish of Paralichthys of Pleuronectiformes such as Scophthalmus maximus, and fish of Cyprinidae such as zebrafish, catfish, eel, Paralichthys, salmon, and tilapia.

Background

The Edwardsiella can cause Edwardsiellosis, is an important fish pathogenic bacterium, can cause diseases to more than 20 kinds of freshwater and seawater fishes such as catfish, turbot, eel and the like, cause gastrointestinal inflammation and hemorrhagic septicemia, cause a great amount of fish death, and seriously threaten the aquaculture industry.

With the continuous expansion of the aquaculture scale of the aquaculture industry, large-scale outbreaks of aquaculture diseases often occur. The annual loss of infectious diseases in fish accounts for more than 20% of the yield, and large-scale outbreaks have become a major obstacle in the aquaculture industry. The main causes of infectious diseases in fish include bacteria, parasites and viruses.

The Edwardsiella can cause Edwardsiellosis, is an important fish pathogenic bacterium, can cause diseases to more than 20 kinds of freshwater and seawater fishes such as catfish, turbot, eel and the like, cause gastrointestinal inflammation and hemorrhagic septicemia, cause a great amount of fish death, and seriously threaten the aquaculture industry. Among them, Edwardsiella piscicola is one of the most toxic species of Edwardsiella.

At present, the large-scale outbreak of the disease is generally controlled by chemically synthesized medicines or antibiotics in mariculture production, but a plurality of disadvantages occur in long-term medication: 1) sometimes, fish diseases have complex etiology, blindness exists when a large amount of medicines are used, and many diseases can not be treated by targeted medicines at present; 2) the chemical drugs have weak preventive effect, and sick fishes have poor food intake and cannot take effective drug dosage; 3) drugs ingested by fish bodies often remain in the fish bodies, which is harmful to human health, and long-term use of the drugs causes environmental pollution and causes the appearance of drug resistance of a large number of microorganisms. Moreover, under the maintenance of long-term medicines, a large number of eliminated individuals with weak disease resistance survive, so that the quality of fish meat is influenced, the disease resistance of the whole population is reduced, and the development of the mariculture industry is seriously influenced.

Therefore, the development of a product or a method with low toxicity or no toxicity, environmental friendliness and ideal effect for preventing and treating diseases in the fish culture process is an urgent problem to be solved in the field. In addition, the industrial characteristics of the aquaculture industry require that the disease control technology must be economical, convenient to apply and implement, so that the products or methods to be developed should also meet the characteristics of economy and practicality.

Disclosure of Invention

The invention aims to provide a novel Edwardsiella attenuated vaccine strain, a preparation method and application thereof.

In a first aspect of the present invention, there is provided an attenuated strain of edwardsiella in which a target gene or a protein encoded by the target gene is down-regulated, the target gene comprising: eseg, EseJ, EseH, ETAE _1586, ETAE _1604, ETAE _2186, ETAE _2188, ETAE _3282, EvpJ.

In a preferred embodiment, the target gene further comprises: rpoS.

In another preferred embodiment, the expression of the esrB gene is also up-regulated in said attenuated strain of Edwardsiella.

In another preferred embodiment, overexpression of esrB gene causes overexpression of T3SS and T6SS secretion system components, thereby promoting immune effect, reducing fish mortality and improving immune protection. After the Edwardsiella attenuated strain is injected into a fish body, T3SS and T6SS secretion system components are expressed in an early excessive manner, but no effector is expressed due to deletion mutation, host immune response is stimulated, an immune protection effect is formed, and the death rate of the Edwardsiella infected by the Edwardsiella attenuated strain is reduced.

In another preferred embodiment, the expression of said esrB gene is driven by a strong promoter; preferably, the strong promoter comprises p_rpsUA promoter.

In another preferred embodiment, the down-regulation comprises: knocking out or silencing a target gene, or inhibiting the activity of a protein encoded by a target gene; preferably, it comprises: knocking out a target gene by a homologous recombination method, knocking out the target gene by a gene editing method, silencing the target gene by a site-specific mutation method, or silencing the target gene by an interference molecule expressed by a specific interference gene; preferably, the homologous recombination method comprises a marker-free in-frame deletion mutation method, such as a marker-free in-frame deletion mutation method based on the suicide plasmid pDMK.

In another preferred embodiment, the edwardsiella includes (but is not limited to): edwardsiella pisciida, Edwardsiella tarda, Edwardsiella ictaluri; preferably, the Edwardsiella piscicida comprises strain EIB 202.

In another preferred embodiment, the attenuated strain is a live bacterial vaccine.

In another preferred example, the EseG Gene (i.e., ETAE _0866) is a Gene having the nucleotide sequence shown in Gene ID: 8607217.

In another preferred example, the EseJ Gene (i.e., ETAE _0888) is a Gene having the nucleotide sequence shown in Gene ID: 8607239.

In another preferred example, the EseH Gene (i.e., ETAE _1757) is a Gene having the nucleotide sequence shown in Gene ID: 8608103.

In another preferred example, the ETAE _1586 Gene is a Gene having a nucleotide sequence shown by Gene ID: 8607934.

In another preferred example, the ETAE _1604 Gene is a Gene having a nucleotide sequence shown by Gene ID: 8607954.

In another preferred example, the ETAE _2186 Gene is a Gene having a nucleotide sequence represented by Gene ID: 8608531.

In another preferred example, the ETAE _2188 Gene is a Gene having a nucleotide sequence represented by Gene ID: 8608533.

In another preferred example, the Gene ETAE _3282 is a Gene having a nucleotide sequence shown by Gene ID: 8609625.

In another preferred example, the EvpJ Gene (i.e., ETAE _2438) is a Gene having the nucleotide sequence shown by Gene ID: 8608783.

In another preferred example, the Rpos Gene is a Gene having a nucleotide sequence shown by Gene ID: 8609217.

In another preferred embodiment, the EsrB gene is a gene having a nucleotide sequence as set forth in GenBank accession FJ 595680.1.

In another preferred embodiment, the attenuated strain has a significantly reduced ability to colonize and adhere to cells as compared to a wild-type strain.

In another aspect of the invention, the use of the attenuated strain of Edwardsiella is provided for preparing a composition for inhibiting Edwardsiella; preferably the composition is a vaccine composition.

In another aspect of the invention, the use of the attenuated strain of edwardsiella is provided for inducing an edwardsiella host to develop immunoprotection against edwardsiella.

In another preferred embodiment, the attenuated strain induces immunoprotection against edwardsiella in fish; preferably, the fish is fish with Edwardsiella as a host; more preferably, the fish species include (but are not limited to): fish of the order flounders, carpiformes, perciformes; more preferably, including (but not limited to): fish of Paralichthys of Pleuronectiformes such as Scophthalmus maximus, and fish of Cyprinidae such as zebrafish, catfish, eel, Paralichthys, salmon, and tilapia.

In another aspect of the invention, a composition is provided, which contains the Edwardsiella attenuated strain and a pharmaceutically acceptable carrier; preferably, the composition is a vaccine composition.

In another aspect of the present invention, there is provided a method of reducing the toxicity of edwardsiella or preparing an attenuated strain of edwardsiella, comprising: down-regulating a target gene or a protein coded by the target gene in a genome of the plant, wherein the target gene comprises: eseg, EseJ, EseH, ETAE _1586, ETAE _1604, ETAE _2186, ETAE _2188, ETAE _3282, EvpJ. In some preferred forms, the target gene further comprises: rpoS. In some further preferred modes, the expression of the esrB gene is also up-regulated in said attenuated strain of edwardsiella.

In another aspect of the invention, there is provided a method (preferably a non-therapeutic method) of increasing the immunoprotection of a fish, comprising: administering to a fish an attenuated strain of edwardsiella, or the composition of claim 8; preferably, the fish is fish with Edwardsiella as a host; more preferably, the fish species include (but are not limited to): fish of the order flounders, carpiformes, perciformes; more preferably, including (but not limited to): fish of Paralichthys of Pleuronectiformes such as Scophthalmus maximus, and fish of Cyprinidae such as zebrafish, catfish, eel, Paralichthys, salmon, and tilapia.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 shows that the infection lethality of the vaccine strain is reduced according to the survival curve of the turbot. In graph A, the injection concentration is 10⁴Each tail of the CFU, FIG. B is 10⁷CFU per tail concentration.

FIG. 2 shows the results of a competition assay (CI) in turbot.

FIG. 3 results of relative immunoprotection (RPS) test

Figure 4, results of in vivo colonization detection.

FIG. 5, map of plasmid pUTT.

Detailed Description

The inventor of the invention has found that targeted down-regulation of a plurality of target genes, better over-expression of a regulatory factor EsrB and/or down-regulation of a virulence inhibiting factor Rpos can kill early and over-expression secretion system components of Edwardsiella multocida, so that a vaccine strain is recognized and eliminated in the early stage of infecting a host fish, and the invention has extremely obvious effects on preventing the Edwardsiella from infecting the host and reducing the infection lethality rate. After the effector is down-regulated, the killing capacity of the Edwardsiella piscicola on a host is reduced, so that the capacity of the Edwardsiella piscicola to infect or colonize the host is reduced.

Effector and secretion component regulation factor, and functional analysis and application thereof

When the pathogenic bacteria interact with the host, various effector proteins can be transported into the host through a secretion system. These virulence proteins can interfere with, modulate and even destroy various pathways and physiological processes of the host, thereby assisting colonization by pathogenic bacteria and causing disease in the host.

The inventor researches and discovers that the obtained vaccine strain which down-regulates EseG, EseJ, EseH, ETAE _1586, ETAE _1604, ETAE _2186, ETAE _2188, ETAE _3282 and EvpJ genes, preferably further up-regulates esrB genes and/or down-regulates Rpos genes can enable the Edwardsiella furacicola to be recognized and eliminated by a host due to early and over-expression of elements but no expression of effectors, thereby playing a role in immune protection. Can obviously reduce the death rate of the fish injected with the vaccine strain caused by the infection of the Edwardsiella ichaeolicus. In a preferred embodiment of the invention, expression of the virulence system is activated by using the PrpsU promoter to drive expression of esrB; more specifically, a pUTat-Prpsu-esrB plasmid vector was established for expression of the esrB gene.

In earlier studies, the inventors found that the function of a response regulator EsrB is not limited to the regulation of virulence, but also participates in other important growth metabolism and environment adaptation related pathways in E.pisciida through transcriptome analysis. Early studies found that the sigma subunit RpoS of RNA polymerase, which is closely related to environmental stress, can act as a repressor of EsrB, and thus represses the expression of EsrB. Subsequent researches find that the Rpos is down-regulated and EsrB is over-expressed, so that secretion systems of T3SS and T6SS are early and over-expressed, the immune effect is achieved, the fish body cannot die, and the vaccine has the characteristic of better immune protection effect compared with the traditional vaccine.

Edwardsiella piscicida has 3T 3SS effector proteins identified, which are analyzed as follows:

EseG (ETAE _0866), which is localized in the host cell membrane and depolymerizes microtubules by amino acids 142-192 of the conserved region; EseJ (ETAE _0888) inhibits pathogen adhesion and aids its intracellular colonization; EseH (ETAE _1757) localizes to the host nucleus and inhibits phosphorylation of the ERK1/2, p38a, and JNK/MAPK pathways in the host. TEM-1 and CyaA reporter systems are used to verify the transport of the above effector proteins into the host cell. EseG (ETAE _0866) is an identified Edwardsiella T3SS protein, and when expressed directly in host cells, EseG can disaggregate the microtubule structure through conserved amino acids 142-192. The EIB202 can form a vesicle containing bacteria (ECVs) in macrophages to survive and proliferate therein, while the EseG is secreted and localized to the vesicle membrane, assisting the stable production of vesicles in an advantageous environment.

EseJ (ETAE _0888), the second identified edwardsiella T3SS effector protein. EseJ inhibits bacterial adhesion to EPC cells, but efficient proliferation of edwardsiella in EPC and j774a.1 is dependent on EseJ. In addition, EseJ can also inhibit the production of Reactive Oxygen Species (ROS) within macrophages to assist edwardsiella in evading host killing.

EseH (ETAE _1757), a phosphoserine enzyme, is highly homologous to shigella OspF, salmonella SpvC, and pseudomonas HopAI 1. OspF is an important virulence factor of shigella T3SS, localizes in the host nucleus, mediates chromatin rearrangement, reduces transcription of inflammatory cytokines, and suppresses the host Innate immune response (lnnate immune response). OspF can also regulate the MAPK pathway of the host and trans-epithelial migration of polymorphonuclear leukocytes, thereby facilitating pathogen infection.

The functions of other 6 proteins than EseG, EseJ and EseH are not clear enough in the prior art.

The inventors analyzed the following:

ETAE _1586, which contains a conserved domain of papain folding toxin; the protein is secreted by a three-type secretion system, can deamidate the 100 th glutamine residue of E2 ubiquitin activating enzyme UBC13 in host cells, and is presumed to be T3SS effector protein inhibiting host inflammatory response through research and analysis by the inventor.

ETAE _1604, which consists of only 49 amino acids and contains a conserved region of extracellular exopeptidase (exortase), was presumed to be involved in secretion, transport, sorting, etc. of proteins and polypeptides by research and analysis.

ETAE _2186, which belongs to Thioredoxin (TRX) family, can change the redox state of a target protein by reversible oxidation of thiol groups, regulate the functions of some enzymes and transcription factors; the present inventors have analyzed their studies and speculated that it may be possible to reduce peroxidases or directly reduce hydrogen peroxide and certain specific free radicals in host cells against oxidative stress defense of host peroxides.

ETAE _2188, which contains a functional domain of DUF1471 of unknown function.

ETAE _3282, which consists of only 39 amino acids, was not found by the present inventors to exist in a conserved domain by identification and analysis.

EvpJ (ETAE _2438) which contains a conserved PAAR domain, the proline-alanine-arginine repeat structure. The inventor has analyzed through research, and speculates that the protein of the family can form a sharp conical structure at the top of a VgrG structure of a six-type secretion system, and the sharp conical structure can assist the secretion and transportation of T6SS effector protein.

The present inventors have analyzed that the above 9 proteins all exhibit T3 SS-dependent characteristics. Of the 9 transporters, EseG, EseH and EseJ have been identified. EseG is specifically localized to the phagosomal membrane and may maintain the stable formation of the vesicle membrane by depolymerizing microtubules, thereby creating a phagosomal space that is conducive to the survival of pathogenic bacteria. EseH is located in the nucleus of host cell, reduces the transcription of cell inflammatory factor and chemotactic factor, and inhibits the ways of MAPK, ERK and the like, thereby inhibiting the inflammatory reaction of host. EseJ is important for the stable proliferation of pathogenic bacteria in a host, and the location and exact target of the pathogenic bacteria in the host cell are not discovered. ETAE _1586 has not yet been identified as a homologous effector protein, and contains a conserved domain of a xylem protease folding toxin which indicates that the conserved domain may have a function of inhibiting an inflammatory response of a host. ETAE _2186 may counteract oxidative stress defense of host peroxides by regulating certain enzymes and or transcription factors. The present inventors believe that these five effector proteins play an important role in assisting pathogenic bacteria in combating the hostile environment of the host and in suppressing the innate immune response of the host. When EIB202 was cultured in vitro, 9 candidate effector protein genes were up-regulated by the EsrB protein. EsrB 202 can induce the transcription of 9 effector genes both in vitro and in vivo in a host. In the early stage of the invention, CI-seq experiments show that EseJ assists EIB202 to fix a field on a host cell, and a plurality of effector proteins of pathogenic bacteria possibly have a synergistic virulence effect. Subsequent fish experiments found that the multiple strain 9 Δ showed relatively significant colonization defects and very weak competition.

In EIB202, EsrB tightly regulated the expression of two systems T3SS/T6 SS. The research of the invention shows that Rpos can regulate the expression of T3SS/T6SS related protein through EsrB. RNA-seq experiments and qRT-PCR technology find that the down regulation of rpoS gene can lead to the collective and significant improvement of the transcription level of genes in T3SS and T6SS gene clusters, which indicates that Rpos can regulate the expression of T3SS/T6SS related protein by regulating the expression of EsrB. RpoS can repress EsrB and its mediated expression of T3SS/T6SS by binding to the EsrB promoter.

In the embodiment of the invention, the inventor knocks out 9 genes for coding effectors, then knocks out the Rpos gene and over-expresses the esrB gene, and successfully constructs 10 delta 8^OEA vaccine strain. The results demonstrate that the 10. delta. esrB^OEAfter the strain is transformed by genetic engineering, the strain can activate T3/T6SS expression early and excessively after entering a host fish body, and is eliminated by host cells, thereby playing a role in immune protection.

In the embodiment of the invention, the inventor directly carries out infection experimental research on fishes, and confirms that the vaccine strain can obviously protect a host from being poisoned by Edwardsiella and can effectively prevent large-scale epidemic diseases caused by Edwardsiella in the aquaculture process.

Based on the new discovery of the inventor, the invention provides the application of effector and secretory component mutant live vaccine strains in preparing a composition for preventing and treating the infection of the Edwardsiella or inducing an Edwardsiella host to generate immune protection against the Edwardsiella. Preferably, the composition is a vaccine composition.

The host associated with the Edwardsiella infection includes marine fish and freshwater fish, and the host includes any fish as the Edwardsiella host. Examples include, but are not limited to: of the order Flounderiformes (including the family Paralichthys), of the order Cypriniformes (including the family Cyprinaceae). More specifically such as but not limited to: turbot of flounder family, zebrafish of carp family, catfish, eel, flounder, salmon, tilapia, etc.

Attenuated strains

The invention provides an attenuated strain of Edwardsiella, wherein a target gene in the attenuated strain is not expressed or is not basically expressed. The target gene comprises: eseg, EseJ, EseH, ETAE _1586, ETAE _1604, ETAE _2186, ETAE _2188, ETAE _3282, EvpJ. Preferably, the target gene further comprises rpoS; preferably, the expression of the esrB gene is also up-regulated in said attenuated strain of Edwardsiella.

In the present invention, the term "substantially no expression" means that the target gene in the attenuated strain of Edwardsiella is not expressed or is expressed at a low level. Wherein the low expression of the target gene means that the expression level of the target gene in the attenuated strain is 30% or lower than that of wild type Edwardsiella; preferably 20% or less than wild type Edwardsiella; more preferably 10% or less of wild type Edwardsiella, most preferably 5% or less, 2% or less of wild type Edwardsiella. The strain in which the target gene is not substantially expressed can be constructed by various techniques such as gene knock-out, gene suppression, gene silencing, gene editing, and the like.

The target gene exists in almost all Edwardsiella species, and the mutation can cause the remarkable reduction of the survival capability of the Edwardsiella in a host body. Due to the conservation of the gene in all Edwardsiella species, the regulation aiming at the gene is applicable to all central Edwardsiella species and has universality.

The target gene also includes gene variants with partial base replacement, and also includes homologues thereof (i.e., genes derived from different species of Edwardsiella but highly homologous (e.g., more than 80%, 85%, 90%, 95%, 98%, 99%) thereof). The target gene also includes truncated variants, so long as the gene fragment is down-regulated (e.g., knocked out) to result in no or aberrant expression of the corresponding target protein, or a reduced or no activity of the target protein fragment expressed thereby. According to the disclosure of the present invention, the target gene or the protein fragment encoded by the target gene can be easily obtained by those skilled in the art.

After knowing the correlation between the target gene and the virulence expression of edwardsiella, various methods well known to those skilled in the art can be used to modulate (down-regulate) the expression of the target gene or the activity of the protein encoded by it. Including but not limited to: (a) knocking out or silencing a target gene in Edwardsiella; (b) transferring a down-regulator for down-regulating a target gene or protein into Edwardsiella; or (c) regulating an upstream signaling pathway or an upstream gene of a target gene in Edwardsiella, to down-regulate target gene expression or protein activity in Edwardsiella.

The downregulation of target gene expression or protein activity in edwardsiella strains can be achieved using a variety of methods known in the art, including but not limited to: gene silencing, gene blocking, gene knockout, gene suppression, and the like. These methods are all included in the present invention.

A preferred method for down-regulating a target gene is gene disruption, in a preferred embodiment of the invention, a target gene disruption plasmid is constructed in vitro, and based on the principle of homologous recombination, the transformation is carried out by a marker-free in-frame deletion mutation method, so that the marker-free in-frame deletion mutation method on the chromosome can no longer encode active proteins. In addition, gene disruption may be performed by deleting a partial region of the gene or inserting an unrelated sequence. The selection of unrelated sequences is readily selectable by the skilled person, for example by the use of resistance genes which may facilitate subsequent selection of strains in which the gene is blocked or knocked out.

In addition, as an alternative operation mode of the invention, the CRISPR/Cas9 system can be adopted for gene editing, so that a target gene is knocked out. Since a suitable sgRNA target site brings higher gene editing efficiency, a suitable target site is designed and found before gene editing is performed. After designing a specific target site, in vitro cell activity screening is also required to obtain an effective target site for subsequent experiments.

The invention also relates to other down-regulating agents (such as antisense nucleic acids, siRNA, miRNA, shRNA, antisense nucleotides and the like) or down-regulating modes of target genes or proteins. Any substance that can inhibit the activity of a target-encoded protein (target protein), down-regulate the stability of the target protein, inhibit the expression of a target gene, reduce the effective acting time of the target protein, or reduce the transcription and translation of the target gene can be used in the present invention as an agent useful for attenuating edwardsiella.

Upregulation of esrB gene expression is also a technique known in the art and can be achieved in a variety of ways. For example, expression systems or expression constructs expressing esrB may be utilized. The expression system or expression construct is not particularly limited, and any expression system that expresses esrB or an analog or mimetic thereof can be used in the present invention. For example, such expression systems or expression constructs include, but are not limited to: an expression vector, a host cell or a virus containing the expression vector. In addition, overexpression of esrB can be further optimized by using strong promoters including p, or inducible promoters, for example, in a preferred embodiment of the invention_rpsUA promoter. In a preferred mode of the present invention, the recombinant expression vector is constructed using pUTT as an initial vector.

In a preferred embodiment of the present invention, an attenuated vaccine strain of edwardsiella is provided as a target unmarked in-frame deletion strain. It has a significantly reduced capacity for colonization, and for adhesion to cells, relative to the wild-type strain. Proved by verification, the vaccine has remarkable immunogenicity, high clearance rate in vivo and very ideal safety and immune response effect.

After entering the body of a host fish, the attenuated vaccine strain of the Edwardsiella provided by the invention can early and excessively express secretion elements of T3SS and T6SS secretion systems, so that the attenuated vaccine strain can be recognized and eliminated by the host in the early infection stage, the mortality rate is low, and the immune protection effect is generated. According to the turbot virulence experiment provided by the embodiment of the invention, compared with a turbot without a vaccine strain, the turbot mortality of the vaccine strain injected is lower than 20%, which shows that the vaccine strain can effectively protect the turbot from being poisoned by Edwardsiella.

After the attenuated vaccine strain of the Edwardsiella of the invention is applied, the host can be obviously protected from being poisoned by the Edwardsiella, and the large-scale epidemic diseases caused by the Edwardsiella in the aquaculture process can be effectively prevented.

Composition comprising a metal oxide and a metal oxide

As used herein, the term "composition of the invention" is generally a pharmaceutical, food or feed composition comprising an attenuated strain of Edwardsiella of the invention (specifically as effector and secretory module mutant live vaccine strain 10. delta. esrB)^OEStrain) as an active ingredient for preventing and treating Edwardsiella infection as a host of Edwardsiella; and a pharmaceutically, dietetically or feedstuffs acceptable carrier or excipient.

In the present invention, the term "comprising" means that various ingredients can be used together in the mixture or composition of the present invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.

In the present invention, a "dietetic or pharmaceutically acceptable" ingredient is a substance that is suitable for use in an animal without undue adverse side effects (such as toxicity, irritation and allergic response) commensurate with a reasonable benefit/risk ratio.

As used herein, the term "Edwardsiella host" refers to an animal, including fish, that can be infected by Edwardsiella (i.e., has an Edwardsiella susceptibility). Preferably, the fish is fish with Edwardsiella as a host; more preferably, the fish species include, but are not limited to: fish of the order flounders, carpiformes, perciformes; more preferably, including but not limited to: fish of Paralichthys of Pleuronectiformes such as Scophthalmus maximus, and fish of Cyprinidae such as zebrafish, catfish, eel, Paralichthys, salmon, and tilapia.

The invention also provides a method for preparing the composition for preventing and treating the Edwardsiella infection, which comprises the step of mixing an effective amount of the composition with a pharmaceutically acceptable carrier to obtain the composition of the invention, wherein the concentration of somatic cells is 10 orders of magnitude²～10⁹CFU/ml, or 10⁶～10⁹CFU/ml。

The dosage form of the composition of the present invention may be various, and any dosage form may be used as long as it can allow the active ingredient to efficiently reach the animal. From the standpoint of ease of preparation and administration, the preferred composition is an oral or injectable formulation. Such as may be selected from: granule, tablet, capsule, solution, suspension, or powder. Various conventional carriers or auxiliary materials required for preparing different dosage forms can be added into the composition, such as fillers, flavoring agents, antioxidants, perfumes, pigments, lubricants, glidants, wetting agents, emulsifiers, pH buffering substances and the like. These additives are well known to those skilled in the art.

In a preferred embodiment of the present invention, the composition is prepared by mixing the attenuated strain of edwardsiella with PBS or physiological saline, and excellent technical effects are achieved in vivo.

The invention also provides a method for preventing and treating the Edwardsiella infection, which comprises the following steps: administering an effective amount of a diluent of a vaccine strain culture solution to a subject (fish) in need thereof. The amount of active ingredient administered is a prophylactically and therapeutically effective amount, which is typically about 100 μ l per fish larvae. Of course, the particular dosage should also take into account factors such as the route of administration.

In one embodiment of the invention, the Edwardsiella tarda vector live vaccine strain or the vaccine composition is administered by injection, wherein the cell concentration of the thallus is 10 orders of magnitude²～10⁹CFU/ml, or 10⁶～10⁹CFU/ml。

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Materials and methods

1. Bacterial species and plasmids

The species and plasmids used in this example are listed in table 1. All strains were stored at-80 ℃ in LB medium containing 20% glycerol. The culture temperature of the Edwardsiella piscicola is 30 ℃, and the culture temperature of the escherichia coli is 37 ℃. When no special indication is given, the rotation speed of the shaking table used for liquid culture is 200 rpm. When needed, antibiotics are added to the medium: polymyxin (polymyxin, Col, 16. mu.g/ml), carbenicillin (Carb, 24. mu.g/ml), kanamycin (kanamycin, Km, 50. mu.g/ml). DMEM medium was purchased from Gibco.

TABLE 1 strains and plasmids

2. Preparation of main reagent and buffer solution

Preparing an antibiotic solution: the antibiotic powder was dissolved in deionized water or absolute ethanol, filter sterilized with a 0.22 μm filter (Millipore, Germany), and dispensed into EP tubes for storage at-20 ℃ for future use. Polymyxin sulfate (PolymyxinB, Inalco), Kanamycin (Kanamycin, Inalco) and carbenicillin (Carbenicilin, Inalco) were dissolved in water, and the concentrations of the stock solutions were 16mg/ml, 50mg/ml and 50mg/ml, and the stock solutions were diluted 1000 times and used as the working concentrations. Chloramphenicol (Chloromycetin, Inalco) is dissolved in absolute ethyl alcohol at a concentration of 75mg/ml mother liquor, and is subpackaged in an EP tube and stored at-20 ℃ for later use. Chloramphenicol was diluted 3000-fold and used as the working concentration. It is noted that chloramphenicol is highly toxic.

LB: weighing the components according to 5g/l of yeast powder, 10g/l of tryptone and 10g/l of NaCl, adding deionized water, adjusting the pH value to 7.0 by using 10mol/l of NaOH, and fixing the volume by using the deionized water. Mixing, and sterilizing with high pressure steam at 121 deg.C for 25 min.

LB solid agar: weighing each component according to 5g/l of yeast powder, 10g/l of tryptone, 10g/l of NaCl and 15g/l of agar, adding deionized water, adjusting the pH value to 7.0 by using 10mol/l of NaOH, and fixing the volume by using the deionized water. Mixing, and sterilizing with high pressure steam at 121 deg.C for 25 min.

10 × TAE buffer: dissolving 242g Tris in baseTo 600ml of double distilled water were added 57.1ml of glacial acetic acid and 37.2g of Na₂EDTA·2H₂O, and metering to 1000 ml. Diluted 10-fold before use.

1.0% agarose gel: weighing 1.0g of agarose, dissolving in 100ml of 1 XTAE buffer solution, heating in a microwave oven for about 2min, pouring into a gel making table quickly after the agarose is completely dissolved, and carrying out spot electrophoresis after cooling for 15 min.

5 × TBE buffer: 54g of Tris base, 27.5g of boric acid and 4.12 g of EDTA are dissolved in 1000ml of ultrapure water.

6% polyacrylamide non-denaturing gel: 9.63ml of ultrapure water, 0.75ml of 50% glycerol, 1.5ml of 5 XTBE buffer, 3ml of 30% AB solution, 0.11ml of 10% APS, 0.01ml of TEMED.

PBS(1L)：8.0g NaCl，0.2g KCl，1.44gNa₂HPO4，0.24g KH₂PO4, add 1L deionized water and mix well.

PBST(1L)：8.0g NaCl，0.2g KCl，1.44g Na₂HPO4，0.24g KH₂PO4, adding 1L deionized water, mixing, adding 500 μ L Tween-20, and mixing.

Sealing liquid: 10% skim milk powder was dissolved in PBST.

Electrotransfer buffer (1L): 3.03g of Tris alkali, 14.4g of glycine and 800ml of deionized water are fully dissolved, 200ml of anhydrous methanol is added and mixed evenly.

3. Strain construction

(1) Construction of in-frame deletion strains

In this example, pDMK plasmid (constructed earlier in the laboratory and constructed by inserting Km resistance gene into pDM4 plasmid) was used. Constructing an in-frame unmarked deletion strain based on the homologous recombination principle. Respectively amplifying fragments of about 500bp upstream and downstream of the target gene, and connecting the fragments to the pDMK plasmid. The R6K replicon of the suicide plasmid pDMK is dependent on the λ pir protein, whereas the EIB202 has no protein encoding this gene and can only maintain its presence by integration into the genome. pDMK enters the EIB202, undergoes a first homologous exchange through a fragment upstream or downstream of the gene of interest, and integrates into the genome. Sac on pDMK^BRThe gene codes levan sucrose transferase, can catalyze sucrose to synthesize levan, and cause the death of EIB202, so that sucrose can be used for preparing foodA second homologous exchange is induced and the plasmid is exchanged.

The pDMK plasmid was extracted and digested with SalI/XbaI in a 37 ℃ water bath for 1 to 1.5 hours. And carrying out DNA agar gel electrophoresis separation on the plasmid subjected to enzyme digestion, selecting a band with the size of 8,700bp, tapping and recovering, and placing the recovered linear DNA at-20 ℃ for later use.

Crude extracting the genome of the strain E.pisciidea EIB202, using the genome as a template, respectively amplifying the upstream and downstream fragments of a target gene by using a corresponding primer pair P1/P2 and a corresponding primer pair P3/P4 which target the specific gene, and purifying and recovering. The upstream and downstream fragments were ligated together by Overlap-PCR using the corresponding P1/P4 primer pair. The ligated upstream and downstream DNA and the digested pDMK were mixed at a molar ratio of 3:1, added to ITA Mix, and subjected to water bath at 50 ℃ for 1 hour, transferred to DH 5. alpha. lamda.pir, and plated on chloramphenicol LB agar plates. The plates were incubated overnight in a 37 ℃ incubator. 12-24 single clones were picked, inoculated with LB, 37 ℃, 200rpm, 2-5h, and verified with the universal primer pair pDMK-F/pDMK-R. Positive clones were inoculated into fresh LB at 37 ℃ and 200rpm overnight. Extracting plasmid and sequencing. The correct plasmid was sequenced, transferred to SM 10. lamda. pir and stored. And performing a conjugation experiment on SM10 lambda pir and EIB202, coating chloramphenicol and polymyxin LB agar plates on the conjugated bacteria liquid, and culturing at 30 ℃ for about 24 hours. Taking 4 monoclonals, verifying single exchange by using a primer pair pDMK-F/Out-R and the primer pair pDMK-R/Out-F, selecting positive, respectively inoculating chloramphenicol and polymyxin LB (lysogeny Broth) for conservation and 12% sucrose LB, inducing for 12h, and carrying Out sacB reverse screening. Diluting the induced bacterial liquid 10³～10⁵12% sucrose LB agar plate was smeared at 30 ℃ for 24 hours. After double exchange, 24-48 monoclonals without fog circles around the monoclonals are picked, and are verified by Out-F/Out-R primers, and the monoclonals with correct band sizes are selected for amplification culture.

TABLE 2 primers

The deletion strain constructed after 9 effector genes (EseG, EseJ, EseH, ETAE _1586, ETAE _1604, ETAE _2186, ETAE _2188, ETAE _3282 and EvpJ) are deleted in sequence is 9 delta, rpoS genes are continuously deleted on the basis, and a 10 delta strain is constructed through sequencing verification.

(2) Construction of EsrB overexpression Strain

In the present invention, an overexpression plasmid is constructed using pUTT plasmid.

The map of plasmid pUTT is shown in FIG. 5. The pUtt plasmid was extracted and digested with restriction endonucleases HindIII/XbaI in a water bath at 37 ℃ for 1 to 1.5 hours. Carrying out DNA agar gel electrophoresis separation on the plasmid after enzyme digestion, selecting a 3584bp strip, cutting the gel and recovering, and placing the recovered linear pUTT plasmid at-20 ℃ for later use.

The prpsU gene is the promoter region of the rpsU gene, rpsU is a subunit of ribosomal protein S21, and is normally expressed, and the promoter is a strong promoter, which the inventors used for overexpression of the esrB gene. Amplifying the prpSU Gene and esrB Gene (amplifying the genome of the suicide Edwardsiella fish, wherein the primers are respectively pUTT-prpSU-F in the positive direction and pUTT-prpSU-R in the reverse direction, pUTT-esrB-F in the positive direction and pUTT-esrB-R in the reverse direction), carrying out DNA gel electrophoresis, mixing the amplified strong promoter prpSU fragment/esrB fragment and the linearized pUTT carrier after enzyme digestion according to the mole number of 3:1, adding ITA Mix (Gibsassambly, Gene Company Limited), carrying out water bath at 50 ℃ for 1 hour, transferring the mixture into DH5 alpha lambda pir and carbenicillin LB agar plates. The plates were incubated overnight in a 37 ℃ incubator. Selecting 12-24 monoclonals, inoculating LB, inoculating at 37 ℃, 200rpm for 2-5h, and verifying by using a universal primer pUTT-F/pUTT-R. Positive clones were inoculated into fresh LB at 37 ℃ and 200rpm overnight. Extracting plasmid and sequencing. Obtaining pUTT-p_rpsUthe-esrB plasmid is extracted and transferred into 10 delta strain by an electrotransformation method to obtain 10 delta esrB^OEAnd (4) strain.

(3) Luciferase assay

Will contain pUtt-p_esrBThe glycerol strain of the deletion strain of the-luxAB plasmid was inoculated into a fresh LB liquid medium and cultured overnight at 37 ℃ and 200 rpm. According to 1% OD₆₀₀Bacterial quantity the strain was inoculated into DMEM medium and cultured by static culture at 28 ℃. At different time points, 200. mu.l of the bacterial solution was added to a 96-well plate with a thoroughly dark edge, and three sets of operations were repeated for each sample. The OD of the 96-well plate was first determined using a Microplate Reader (Bio-Tek)₆₀₀The absorbance was measured by adding 40. mu.l of 1% decanal (dissolved in pure alcohol) as a substrate to a chemiluminescence apparatus (Microplate Luminometer) to measure the luciferase expression level.

(4) Turbot in vivo toxicity test

EIB202 wild strain, 10. delta. strain prepared as described above and 10. delta. esrB^OEThe strain was inoculated in LB medium, cultured overnight, and second-stage inoculated in DMEM, and cultured at 28 ℃ under static conditions. The cells were collected by centrifugation at 5000g for 4min, washed 3 times with sterile PBS, and diluted to 100 CFU/. mu.l each. 100 μ l of diluted bacterial solution was injected intramuscularly to each fish, while zebrafish injected 100 μ l of PBS per tail muscle was used as a control. Each group is provided with 15 fishes. And observing and recording the morbidity and the mortality of the experimental group and the control group every 5 hours after the challenge, and timely cleaning dead fish and changing water. And finally, counting the survival and death conditions of each group of fishes, and drawing a survival curve graph.

(5) In vivo colonization detection

Taking EIB202 wild strain (WT), 9 delta strain, 10 delta strain and 10 delta esrB^OEInoculating LB culture medium to the strain, culturing overnight, inoculating DMEM to the strain, and statically culturing at 28 deg.C, wherein the culture solution is used for performing challenge experiment on about 30g turbot. Each group is provided with 15 fishes, and the number of the fishes is 3 in parallel. Weighing, grinding, coating and counting livers at 4 th, 7 th and 11 th days after the toxin attack, wherein the final relative planting rate calculation formula is as follows:

the relative colonization rate = (actual viable count CFU of experimental group/liver mass μ g of experimental group)/(actual viable count CFU of control group/liver mass μ g of control group) × 100%.

(6) Turbot in vivo competitive assay (CI)

Collecting 1ml of related Edwardsiella strain secondary strain cultured at 37 deg.C overnight with shaking, centrifuging at 5000 × g for 2min, collecting thallus, washing thallus with sterile PBSWashed 3 times and then diluted to 5X 10⁵CFU/ml, EIB202 strains were mixed at a ratio of 1:1 as required. The mixed bacterial solution was injected into each fish in an amount of 100. mu.l for each group of 15 fish. On the fifth day after challenge (DPI ═ 5), 5 fish livers were individually ground and diluted in gradient plates (individually plated onto screening-resistant and blank DHL plates). The cells were incubated at 37 ℃ overnight and colony counts were performed.

(7) Relative immunoprotection (RPS) assay

Let WT, 10 Δ esrB^OECentrifuging the first and second culture fluid at 5000rpm for 3min to collect thallus, washing thallus with sterile PBS for 3 times, and diluting to 10⁶CFU/ml. 100 μ l of diluted bacterial suspension was injected into each fish via intraperitoneal injection, and the fish injected with 100 μ l of PBS per tail muscle was used as a control, and 120 fish were used for each group. After immunization, the plants are cultured normally in the system. Collecting blood 35 days after immunization, inserting a syringe into spinal blood vessels from the tail muscle of the back for blood drawing, taking 3 fish in each group, standing the blood at 4 ℃ overnight, collecting serum the next day, storing at-80 ℃, and then determining the antibody titer.

The wild strains were challenged 35 days after immunization and the relative immunoprotection was assessed. Centrifuging at 5000r/min for 3min to collect thallus, washing thallus with sterile PBS for 3 times, and diluting to 2 × 10⁴CFU/ml. 100 μ l of diluted bacterial solution was injected intramuscularly to each fish of the PBS control group and 3 immunization groups, each group being 3 replicates, each replicate 30 fish. After the toxin is attacked, the disease and death conditions of the experimental group and the control group are recorded every day after 28 days of observation, and the dead fish are cleaned in time and changed with water. Finally, counting the survival and death conditions of each group of fishes, drawing a survival curve, and calculating the relative immune protective power according to the following formula:

RPS ═ (1-% mortality in the immunization group/mortality in the PBS control group) × 100%.

Example 1 virulence test experiment Using turbot as test animal

Are respectively 1 × 10⁴CFU/Tail and 1X 10⁷The immune healthy turbot is injected into the abdominal cavity by CFU/tail dosage, the turbot used in the test is randomly divided into 4 groups, each group comprises 3 parallel water tanks and 15 tails/tank. The prepared toxin is attenuatedLive vaccines and the like are immunized by an intraperitoneal injection mode. Experimental groups injected with wild Edwardsiella EIB202 strain (WT), 10 gene-deleted Edwardsiella strain (10 Delta), and esrB strain overexpressed after Edwardsiella 10 deletion (10 Delta esrB)^OE) The control group was injected with an equal volume of sterile PBS buffer. The immunization period was 20 days, and the health of the experimental animals was observed daily and survival and death were recorded.

As shown in FIG. 1, at 14 days of injection, 1X 10⁴The WT group of CFU/tail was close to 70% dead, 10 Δ and 10 Δ esrB^OENo fish died; at the same time, 1 × 10⁷The CFU/tailed WT group died entirely on day 14, with mortality rates of 40% and 35% for the 10 Δ esrBOE groups, respectively. At 20 days, 1X 10⁴The CFU/tailed WT groups had all died, 10 Δ and 10 Δ esrB^OEGroup mortality was 36% and 42%, respectively; 1X 10⁷CFU/tail 10 Δ and 10 Δ esrB^OEGroup mortality was 52% and 42%, respectively.

Thus, 10 Δ and 10 Δ esrB of the present invention^OEThe toxicity of the vaccine group strain to the turbot is greatly reduced compared with the wild type.

Example 2 validation of animal level

1. Turbot in vivo competitive assay (CI)

Collecting 1ml of related Edwardsiella strain secondary strain cultured at 37 deg.C overnight with shaking, centrifuging at 5000 × g for 2min to collect thallus, washing thallus with sterile PBS for 3 times, and diluting to 5 × 10⁵CFU/ml, EIB202 strains were mixed at a ratio of 1:1 as required. The mixed bacterial solution was injected into each fish in an amount of 100. mu.l for each group of 15 fish. On day 5 after challenge (DPI ═ 5), 5 fish livers were individually ground and diluted in gradient plates (plated onto screening-resistant and blank DHL plates). The cells were incubated at 37 ℃ overnight and colony counts were performed.

As a result, as shown in FIG. 2, in vivo competition experiments were carried out using EIB 202. delta. p strain as a control, and 10. delta. esrB were found^OEThe competitive power of the strain in the turbot body is obviously lower than that of an EIB202 wild strain (WT), and the overexpression of esrB gene indicates that effector is deleted and T3SS and T6SS modules are activated to express, so that the host can be enabled to infect earlyThis strain is recognized and subsequently eliminated, producing an immunoprotective effect. Not only reducing the lethality of vaccine strains, but also improving the immune protection.

(2) Test for relative immunoprotection (RPS) assay

EIB202 wild strain (WT), 9. delta. strain, 10. delta. esrB^OECentrifuging the first and second culture fluid at 5000rpm for 3min to collect thallus, washing thallus with sterile PBS for 3 times, and diluting to 10⁶CFU/ml. 100 μ l of diluted bacterial suspension was injected into each fish via intraperitoneal injection, and the fish injected with 100 μ l of PBS into each tail muscle was used as a control, and 120 fish were used for each group. After immunization, the plants are cultured normally in the system. Collecting blood 35 days after immunization, inserting a syringe into spinal blood vessels from the tail muscle of the back for blood drawing, taking 3 fish in each group, standing the blood at 4 ℃ overnight, collecting serum the next day, storing at-80 ℃, and then determining the antibody titer.

The wild strains were challenged 35 days after immunization and the relative immunoprotection was assessed. Centrifuging at 5000r/min for 3min to collect thallus, washing thallus with sterile PBS for 3 times, and diluting to 2 × 10⁴CFU/ml. 100 μ l of diluted bacterial solution was injected intramuscularly to each fish of the PBS control group and 3 immunization groups, each group being 3 replicates, each replicate 30 fish. After the toxin is attacked, the disease and death conditions of the experimental group and the control group are recorded every day after 28 days of observation, and the dead fish are cleaned in time and changed with water.

Finally, counting the survival and death conditions of each group of fishes, drawing a survival curve, and calculating the relative immune protective power according to the following formula:

RPS ═ (1-% mortality in the immunization group/mortality in the PBS control group) × 100%.

As shown in FIG. 3, 9. delta. strains, 10. delta. strains and 10. delta. esrB^OEThe strains have higher immune protection after injection, wherein 10 delta esrB is used^OEIs the highest. The vaccine strain can generate good immune protection effect on the host fish body after being inoculated.

(3) In vivo field planting detection experiment

Taking EIB202 wild strain, 9 delta strain, 10 delta strain and 10 delta esrB^OEInoculating strain to LB medium, culturing overnight, inoculating DMEM to the second stage, and standing at 28 deg.C, wherein the culture solution is used for 30gPerforming a toxicity attacking experiment on the right turbot. Each group is provided with 15 fishes, and the number of the fishes is 3 in parallel. Weighing, grinding, coating and counting livers at 4 th, 7 th and 11 th days after the toxin attack, wherein the final relative planting rate calculation formula is as follows:

the relative colonization rate = (actual viable count CFU of experimental group/liver mass μ g of experimental group)/(actual viable count CFU of control group/liver mass μ g of control group) × 100%.

As can be seen from the results in FIG. 4, 9. delta. strains, 10. delta. strains and 10. delta. esrB at day 4^OEThe planting rate of the plants is not greatly different from that of wild plants. However, at day 7, the rates of colonization of these three plants relative to the wild type were significantly reduced, indicating that the deletion was recognized and cleared in the host, and of these 10. delta. esrB^OEThe most obvious situation is when the strain is eliminated.

Therefore, the administration of effector and secretory component mutant live vaccine strains to host fish very significantly protects fish from damage by edwardsiella.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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<211> 20

<212> DNA

<213> primers (Primer)

<400> 59

gccatcgtag tagggcaaca 20

<210> 60

<211> 20

<212> DNA

<213> primers (Primer)

<400> 60

gacgatggac aggccgttat 20

<210> 61

<211> 43

<212> DNA

<213> primers (Primer)

<400> 61

gagctcaggt tacccggatc tatgagctat aacggcgaag cct 43

<210> 62

<211> 30

<212> DNA

<213> primers (Primer)

<400> 62

gcatcgctca catagctgta ccctacccgt 30

<210> 63

<211> 30

<212> DNA

<213> primers (Primer)

<400> 63

tacagctatg tgagcgatgc ggtcaaaaaa 30

<210> 64

<211> 48

<212> DNA

<213> primers (Primer)

<400> 64

ccctcgagta cgcgtcacta gtggggccct ctgttcgcca cccctact 48

<210> 65

<211> 20

<212> DNA

<213> primers (Primer)

<400> 65

ataaccgcag ctaccagtcg 20

<210> 66

<211> 20

<212> DNA

<213> primers (Primer)

<400> 66

cccggtcgat cgcatcttta 20

<210> 67

<211> 21

<212> DNA

<213> primers (Primer)

<400> 67

aaagctctca tcaaccgtgg c 21

<210> 68

<211> 21

<212> DNA

<213> primers (Primer)

<400> 68

tgctccagtg gcttctgttt c 21

<210> 69

<211> 45

<212> DNA

<213> primers (Primer)

<400> 69

aagatggcgc agcagggcgt ggggaattcc tccttaattt ttaac 45

<210> 70

<211> 49

<212> DNA

<213> primers (Primer)

<400> 70

gtctccgttt tatctttatc agtacacgga gagtggagct atttttagc 49

<210> 71

<211> 49

<212> DNA

<213> primers (Primer)

<400> 71

cgatttttat cgaggtgaga ggcacatgac tatttctatt ttgcctctg 49

<210> 72

<211> 42

<212> DNA

<213> primers (Primer)

<400> 72

tctcatccgc caaaacagcc ttaaaactcc agaaccccca gg 42