KR20190046731A - Pharmaceutical composition for preventing or treating edwardsiellosis of fish, and a method for preventing or treating edwardsiellosis of fish - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating edwardsiellosis of fish, and a method for preventing and treating edwardsiellosis of fish. The pharmaceutical composition comprises the nucleic acid molecule of SEQ ID NO: 1 as an active ingredient, wherein the SEQ ID NO: 1 is mixed with a recombinant lactococcus lactis strain.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a pharmaceutical composition for preventing or treating Edward's disease in fish, and a method for preventing and treating Edward's disease in fish,

The present invention relates to a pharmaceutical composition for the prevention or treatment of Edwards disease of fish, and a method for preventing and treating Edward's disease in fish.

Edwardsiella tarda ) is a rodent, facultatively anaerobic, and gram-negative pathogen that causes edwardsiellosis in many animals, especially fish [BR Mohanty and PK Sahoo, Edwardsiellosis in fish: a brief review, J Biosci. 32 (2007) 1331-1334.], [T. Xu and X.-H. Zhang, Edwardsiella tarda : an intriguing problem in aquaculture, Aquaculture 431 (2014) 129-135.]. The importance of controlling Edward's disease has been emphasized because of the enormous damage done at the farm [T. Xu and X.-H. Zhang, Edwardsiella tarda : an intriguing problem in aquaculture, Aquaculture 431 (2014) 129-135.]. E. tarda at the farm Over the past several decades of studying how to combat infection, E. tarda has been exposed to formalin-sagittal cells [M. Yamasaki, K. Araki, K. Maruyoshi, M. Matsumoto, C. Nakayasu, T. Morimoto, et al., Comparative analysis of adaptive immune response after vaccine trials using live ateenuated and formalin- killed cells of Edwardsiella tarda in ginbuna crucian carp (Carassius auratus langsdorfii), Fish Shellfish Immunol. 45 (2015) 437-442.], [N. Kaneshige, W. Jirapongpairoj, I. Hirono, H. Kondo, Temperature-dependent regulation of gene expression in Japanese flounder Paralichthys olivaceus kidney after Edwardsiella tarda formalin-killed cells. Fish Shellfish Immunol. 59 (2016) 298-304.], Ghost vaccine [SR Kwon, YK Nam, SK Kim, KH Kim, Protection of tilapia ( Oreochromis mosambicus ) from edwardsiellosis by vaccination with Edwardsiella tarda ghosts, Fish Shellfish Immunol. 20 (2006) 621-626.], [SR Kwon, EH Lee, YK Nam, SK Kim, KH Kim, Efficacy of oral immunization with Edwardsiella tarda ghosts against edwardsiellosis in olive flounder ( Paralichthys olivaceu s), Aquaculture 269 (2007 ) 84-88.], [DJ Lee, SR Kwon, K. Zenke, EH Lee, YK Nam, SK Kim et al., Generation of safety enhanced Edwardsiella tarda ghost vaccine, Dis. Aquat. Org. 81 (2008) 249-254.], An attenuated mutant strain [SH Choi, KH Kim, Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder ( Paralichthys olivaceus ). Fish Shellfish Immunol. 31 (2011) 58-65.], [J. Xiao, T. Chen, B. Liu, W. Yang, Q. Wang, J. Qu, et al., Edwardsiella tarda mutant disrupted type III secretion system and chorismic acid synthesis and cured of a plasmid as a live attenuated vaccine in turbot, Fish Shellfish Immunol. 35 (2013) 632-641.], [J. Li, Z. Mo, G. Li, P. Xiao, J. Huang, Generation and evaluation of virulence attenuated mutants of Edwardsiella tarda as vaccine candidates to combat edwradsiellosis in flounder ( Paralichthys olivaceus ), Fish Shellfish Immunol. 43 (2015) 175-180.], And DNA vaccines [Y. Sun, C.-S. Liu, L. Sun, Comparative study of the immune effect of an Edwardsiella tarda antigen in two forms: Subunit vaccine vs DNA vaccine, Vaccine 29 (2011) 2051-2057.], [X.-d. Jiao, M. Zhang, Y.-h. Hu, L. Sun, Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens, Vaccine 27 (2009) 5195-5202.], [X. Liu, J. Xu, H. Zhang, Q. Liu, J. Xiao, Y. Zhang, Design and evaluation of an Edwardsiella tarda DNA vaccine co-encoding antigenic and adjuvant peptide, Fish Shellfish Immunol. 59 (2016) 189-195.], [F. Liu, X. Tang, X. Sheng, J. Xing, W. Zhan, DNA vaccine encoding molecular chaperone, GroEL of Edwardsiella tarda confers protective efficacy against edwardsiellosis, Mol. Immunol. 79 (2016) 55-65.] Has been extensively studied as a disease prevention strategy.

Among them, the vaccine strategy is a basic and efficient approach to control infectious diseases, because the mechanism is based on bringing up the adaptive immune system and creating memory in the host's immune system against pathogens. In addition, the use of vaccines can greatly reduce the use of antibiotics in animal breeding sites for diseases caused by bacterial infections.

While many types of vaccines are being studied, the delivery system and route of vaccination is a major issue in vaccine strategies. In particular, in the aquaculture field, conventional vaccine-type vaccines have some disadvantages: a large amount of labor due to the mass culture of fish, secondary infections due to needle punctures, and handling stress of fish [K.P. Plant, S.E. LaPatra, Advances in fish vaccine delivery, Dev. Comp. Immunol. 35 (2011) 1256-1262.], [B.E. Brudeseth, R. Wiulsrod, B.N. Fredriksen, K. Lindmo, K.E. Lokling, M. Bordevik, et al., Status and future perspectives of vaccines for industrialized fin-fish farming, Fish Shellfish Immunol. 35 (2013) 1759-1768.]. In order to avoid these drawbacks, oral administration, in particular feed vaccination, is an ideal method for vaccination against fish, since feeding is an essential step in fish culture.

From the point of view of the delivery system, the recombinant Lactococcus lactis BFE920, a vaccine vehicle herein, Kim, BR Beck, S.-B. Heo, J. Kim, HD Kim, S.-M. Lee et al., Lactococcus lactis BFE920 activates the innate immune system of olive flounder ( Paralichthys olivaceus ), resulting in protection against Streptococcus iniae infection and enhancing feed efficiency and weight gain in large-scale field studies, Fish Shellfish Immunol. 35 (2013) 1585-1590.], [BR Beck, D. Kim, J. Jeon, S.-M. Lee, HK Kim, O.-J. Kim, et al., The effects of combined dietary probiotics Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 on innate immunity and disease resistance in olive flounder ( Paralichthys olivaceus ), Fish Shellfish Immunol. 42 (2015) 177-183.], [BR Beck, JH Song, BS Park, D. Kim, J.-H. Kwak, HK Do, et al., Distinct immune tones are established by Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 in the gut of olive flounder ( Paralichthys olivaceus ), Fish Shellfish Immunol. 55 (2016) 434-443.) Is a grade of lactic acid bacteria (LAB) that is proven to be probiotic and generally regarded as safe. In addition, L. lactis species have been reported in a number of studies in both mouse and fish systems, including L. lactis BFE920 [K. Robinson, LM Chamberlain, KM Schofield, JM Wells, RWF Le Page, Oral vaccination of mice with tetanus with recombinant Lactococcus lactis . Nat. Biotechnol. 15 (1997) 653-657. [MH Lee, Y. Roussel, M. Wilks, S. Tabaqchali, Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H. pylori infection in mice, Vaccine 19 (2001) 3927-3935. Wang, L. Huang, J. Kong, X. Zhang, Expression of the capsid protein of porcine circovirus type 2 in Lactococcus lactis for oral vaccination, J. Virol. Methods 150 (2008) 1-6. Anuradha, HL Foo, NS Mariana, TC Loh, K. Yusoff, MD Hassan, et al., Live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 and D4 for protection against Aeromonas hydrophila in tilapia ( Oreochromis niloticus ), J. Appl. Microbiol. 109 (2010) 1632-1642. Thus, the safety and probiotic effects of L. lactis BFE920 make it a viable and interesting vaccine delivery system to achieve successful feed vaccination methods. Taken together, feed vaccination of recombinant L. lactis BFE920 is a promising vaccine technology in the field.

Recently, recombinant vaccine technology has been widely used and studied as mentioned above [K. Kawai, Y. Liu, K. Ohnishi, S.-i. Oshima, A conserved 37 kDa outer membrane protein of Edwardsiella tarda is an effective vaccine candidate, Vaccine 22 (2004) 3411-3418. Sun, C.-S. Liu, L. Sun, Comparative study of the immune effect of an Edwardsiella tarda antigen in two forms: Subunit vaccine vs DNA vaccine, Vaccine 29 (2011) 2051-2057. Maiti, M. Shetty, M. Shekar, I. Karunasagar, I. Karunasagar, Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda , a potential vaccine candidate for fish, common carp. Microbiol. Res. 167 (2011) 1-7.], [M. Zhang, H. Wu, X. Li, M. Yang, T. Chen, Q. Wang, et al., Edwardsiella tarda flagellar protein FlgD: A protective immunogen against edwardsiellosis, Vaccine 30 (2012) 3849-3856.], [X.-d. Jiao, M. Zhang, Y.-h. Hu, L. Sun, Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens, Vaccine 27 (2009) 5195-5202.], [X. Liu, J. Xu, H. Zhang, Q. Liu, J. Xiao, Y. Zhang, Design and evaluation of an Edwardsiella tarda DNA vaccine co-encoding antigenic and adjuvant peptide, Fish Shellfish Immunol. 59 (2016) 189-195.], [F. Liu, X. Tang, X. Sheng, J. Xing, W. Zhan, DNA vaccine encoding molecular chaperone GroEL of Edwardsiella tarda confers protective efficacy against edwardsiellosis, Mol. Immunol. 79 (2016) 55-65.]. Recombinant DNA / protein subunit vaccines have certain desirable advantages, such as desired target specificity, and further, antigen modification for efficiency, and safety, but an adjuvant is required to elicit an appropriate vaccine effect [M. Hansson, P.-Å. Nygren, S. Stahl, Design and production of recombinant subunit vaccines, Biotechnol. Appl. Biochem. 32 (2000) 95-107.], [TG Clark, D. Cassidy-Hanley, Recombinant subunit vaccines: Potentials and constraints, Dev. Biol. 121 (2005) 153-163.]. To overcome these drawbacks, L. lactis BFE920 has a congenital immune stimulating effect through induction of IFN- gamma by the host L. lactis BFE920, and SiMA of Streptococcus iniae has been reported in a previous study [D. Kim, BR Beck, SM Lee, J. Jeon, DW Lee, JI Lee, et al., Pellet feed adsorbed with the recombinant Lactococcus lactis BFE920 expressing SiMA antigen against strong recall vaccine against Streptococcus iniae infection in olive flounder ( Paralichthys olivaceus ), Fish Shellfish Immunol. 55 (2016) 374-383.), The vaccine effect can be applied as a solution.

From E. tarda antigen, outer membrane protein of E. tarda (E. tarda outer membrane protein, Omp) [K. Kawai, Y. Liu, K. Ohnishi, S.-i. Oshima, A conserved 37 kDa outer membrane protein of Edwardsiella tarde is an effective vaccine candidate, Vaccine 22 (2004) 3411-3418. Maiti, M. Shetty, M. Shekar, I. Karunasagar, I. Karunasagar, Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda , a potential vaccine candidate for fish, common carp. Microbiol. Res. 167 (2011) 1-7] and flagellar hook protein D, FlgD [M. Zhang, H. Wu, X. Li, M. Yang, T. Chen, Q. Wang, et al., Edwardsiella tarda flagellar protein FlgD: A protective immunogen against edwardsiellosis, Vaccine 30 (2012) 3849-3856. X. Liu, J. Xu, H. Zhang, Q. Liu, J. Xiao, Y. Zhang, Design and evaluation of an Edwardsiella tarda DNA vaccine co-encoding antigenic and adjuvant peptide, Fish Shellfish Immunol. 59 (2016) 189-195.] Showed a vaccine effect in a fish model. Thus, the combination of information of the given vehicle and antigens can provide a feed animal vaccine model for Edwards disease on the diet.

Also, in order to increase the epitope diversity of the antigen and the vaccine efficacy, the flexible glycine / serine (G / S) linker [X. Chen, J. L. Zaro, W.-C. Shen, Fusion protein linkers: Property, design and functionality, Adv. Drug Deliv. Rev. 65 (2013) 1357-1369. The fusion protein of OmpA and FlgD was designed.

The present invention relates to a pharmaceutical composition for the prevention or treatment of Edwards disease of fish comprising Edwardian elastase outer membrane protein A (OmpA), flagella hook protein D (FlgD), flexible glycine / serine (G / S) Recombinant Lactococcus lactis BFE920 ( L. lactis BFE920) expressing a fusion protein of an antigen that binds to E. coli was developed. By utilizing the vaccine effect on T cells, pathogen-specific antibody production, and protective effect against E. tarda , And to provide a method for the prevention and treatment of Edwards disease in fish.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

According to a first aspect of the present invention, there is provided a vaccine composition for immunomodulation comprising the nucleic acid molecule of SEQ ID NO: 1 as an active ingredient.

The second aspect of the present invention provides a pharmaceutical composition for preventing or treating Edward's disease in fish, comprising recombinant Lactococcus lactis BFE920 strain (Accession No. KCCM11383P) as an active ingredient.

A third aspect of the invention provides a method of preventing or treating Edwards disease in a fish, comprising the step of administering to the fish the pharmaceutical composition comprising the recombinant Lactococcus lactis BFE920 (accession number: KCCM11383P).

The fourth aspect of the present invention provides a feed composition for preventing or alleviating Edwards disease in fish, comprising recombinant Lactococcus lactis BFE920 (accession number: KCCM11383P) as an active ingredient.

The fifth aspect of the present invention provides a feed additive composition for preventing or improving Edwards disease of fish, which comprises recombinant Lactococcus lactis strain BFE920 (accession number: KCCM11383P) as an active ingredient.

According to one embodiment of the present disclosure, A recombinant Lactococcus lactis BFE920 strain deposited with the deposit number KCCM11383P expressing a fusion antigen consisting of the above two species, or an outer membrane protein A ( Edwardsiella tarda outer membrane protein, OmpA), flagellar hook protein D, FlgD Can be developed.

According to one embodiment of the present invention, the recombinant Lactococcus lactis strain BFE920 may be a nucleic acid molecule of SEQ ID NO: 1 incorporated therein.

According to one embodiment of the present invention, the vaccine composition for immunity enhancement comprising the recombinant Lactococcus lactis BFE920 strain deposited with the deposit number KCCM11383P can be used as a composition comprising the wild-type Lactococcus lactis BFE920 strain, a composition comprising OmpA or FlgD Can exhibit significantly improved T cell and Th1 subset responses and adjuvant effects against FlgD, as compared to compositions for the prevention or treatment of Edwards disease of fish expressing only a single antigen, Can increase the survival rate of fish, especially flounder, due to infection of the flounder.

The pharmaceutical composition for preventing or treating Edward's disease according to one embodiment of the present invention is a vaccine for preventing or treating Edward's disease that is safe and probiotic effect of the vehicle and also includes a vaccine containing wild-type Lactococcus lactis BFE920, OmpA Or FlgD as compared to a pharmaceutical composition for the prevention or treatment of Edwards disease of fish expressing only one of the single antigens of FlgD or FlgD, it is possible to provide a promising vaccine for the prevention and treatment of Edwards disease of fish, It can be a candidate material.

1 is a simplified schematic diagram of a feed vaccination protocol in one embodiment of the present invention. Samples were collected on the last day of each vaccination week and during the rest period, the usual feed was fed to all experimental groups.
Figs. 2 (a) to 2 (c) are schematic diagrams showing a plasmid production step in one embodiment of the present invention. Fig. Figure 1 (a) shows the antigenic DNA insert of each vaccine strain, (b) shows the PCR cycle used for DNA insert amplification and (c) shows a brief description of the plasmid information. Same as: Cm ^R , chloramphenicol resistance gene; PnisA, Nysin promoter; RepA, plasmid replication protein A; RepC, plasmid replication protein C].
Figure 3 shows the total cell lysate analyzed by western blot analysis under denaturing conditions, in one embodiment of the invention.
Figures 4 (a) - (f) show that in one embodiment of the present invention, the increased T cell response ( CD4-1, CD4-2) by the treatment of the recombinant L. lactis BFE920 E. tarda vaccine strain in the flounder of flounder , And CD8? ). In Figure 4, CD4-1, CD4-2, and < RTI ID = 0.0 &gt; The CD8α responses are shown in (a) to (c), while the CD4-1, CD4-2, and CD8α during the boost vaccination are shown in (d) to (f). Statistical significance was as follows (for the control group, p <0.05, ** p <0.001, and *** represents p <0.0001; for WT LL, + p <0.05, ++ p < 0.001, and +++ indicates p < 0.0001).
5 (a) to 5 (d) are graphs showing the results of an up-regulated Th1 subset reaction ( T-bet and T-bet) according to the treatment of the flounder intestine recombinant L. lactis BFE920 E. tarda vaccine strain IFN- ? ). In FIG. 5, T-bet and IFN- gamma responses during prime vaccination are shown in (a) and (b), while T-bet and IFN- gamma responses during boost vaccination are shown in (c) and (d). Statistical significance was as follows (for the control group, p <0.05, ** p <0.001, and *** represents p <0.0001; for WT LL, + p <0.05, ++ p < 0.001, and +++ indicates p < 0.0001).
Figures 6 (a) - (c) show that in one embodiment of the invention TLR5M (a), IL-l [beta] (b), and IL- 12p40 (c) reaction. Statistical significance was as follows (for the control group, p <0.05, ** p <0.001, and *** represents p <0.0001; for WT LL, + p <0.05, ++ p < 0.001, and +++ indicates p < 0.0001).
Figures 7 (a) and 7 (b) are graphs showing the E. tarda specific antibody response of the flounder detected during prime vaccination (a) and during boost vaccination (b), in one embodiment of the invention . Statistical significance was as follows (for the control group, p <0.05, ** p <0.001, and *** represents p <0.0001; for WT LL, + p <0.05, ++ p < 0.001, and +++ indicates p < 0.0001).
8 is a graph comparing the log-rank method (Mantel-Cox) of the control (Ctrl) and L. lactis BFE920 group (WT LL) in one embodiment of the present invention.
9 (a) and 9 (b) are graphs showing changes in body weight gain (PWG) in the control group and the experimental group, and (b) in the feed conversion ratio (FCR). Six repeated measurements of 20 randomly selected fish were presented with standard error (SEM) (* indicates p < 0.05).
10 is a graph showing the mean of three trials of four individual fish in the control (Ctrl), WT LL, FlgD, OmpA, and Fusion groups, along with standard error (SEM) (* Represents p &lt; 0.05 and ** represents p &lt; 0.001).

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In the drawings, the same reference numbers are used throughout the specification to refer to the same or like parts.

Throughout this specification, when a part is referred to as being "connected" to another part, it is not limited to a case where it is "directly connected" but also includes the case where it is "electrically connected" do.

Throughout this specification, when a member is &quot; on &quot; another member, it includes not only when the member is in contact with the other member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as &quot; including &quot; an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise. The terms &quot; about &quot;, &quot; substantially &quot;, etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word &quot; step (or step) &quot; or &quot; step &quot; used to the extent that it is used throughout the specification does not mean &quot; step for.

Throughout this specification, the term &quot; combination (s) thereof &quot; included in the expression of the machine form means a mixture or combination of one or more elements selected from the group consisting of the constituents described in the expression of the form of a marker, Quot; means at least one selected from the group consisting of the above-mentioned elements.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Throughout the present specification, "Edward bottle (Edawardsiellosis)" of the substrate is Edward when Ella tar belonging to the family Enterobacteriaceae (Edwardsiella tarda , E. tarda ) It means fish disease caused by bacteria. It occurs mainly from high-temperature early summer to autumn and does not induce mass mortality in a short time, but it can lead to long-term mortality. The above-mentioned Edwards disease can cause a great economic loss to the aquaculture industry of the flounder, which is the most important species in Korean aquaculture. Edwards disease infected flounder is a characteristic symptom of abdominal distension and hernia. The blood is mixed with ascites, the oily skin is observed, the liver is brown and the ovary is formed, and the liver and kidney are hypertrophied and may carry meningitis have.

Throughout the present specification, the description of " Lactococcus " means lactic acid (lactic acid) bacteria belonging to the genus Streptococcus Group N1, also called lactic acid bacteria. It is known as the main or unique product of glucose fermentation, specifically a homofermentor that produces lactic acid. The strain may be widely used in the dairy industry to produce fermented milk products such as cheese, yogurt and the like.

Throughout this specification, the term " Lactococcus lactis " lactis , L. lactis ) is one of the lactic acid bacteria belonging to the above-mentioned Lactococcus species and can be used for the production of industrial dairy products such as cheese, fermented milk or lactic casein and can be used as a probiotic.

Throughout the present specification, the description of " anti-inflammation " means an action to suppress inflammation, and the description of " immunity enhancement " refers to all phenomena that reinforce the body defense mechanism against various diseases such as cancer, inflammation and the like in vivo .

Throughout this specification, the term " feed " means any natural or artificial diet, single meal or the like or ingredients of the above formula for eating, ingesting, digesting or suitable for the subject organism.

Throughout the present specification, "feed additive" means a substance added to the feed to improve the productivity or health of the target organism.

Throughout this specification, the phrase " administering " means introducing a predetermined substance into a subject in any suitable manner, and the administration route of the composition can be administered through any conventional route so long as it can reach the target tissue . For example, the substance can be administered to a subject intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intracisternally, or rectally, But may not be limited.

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to these embodiments and examples and drawings.

According to a first aspect of the present invention, there is provided a vaccine composition for immunomodulation comprising the nucleic acid molecule of SEQ ID NO: 1 as an active ingredient.

In one embodiment herein, the sequence 1 may be incorporated into a recombinant Lactococcus lactis strain. For example, the sequence 1 may encode SEQ ID NO: 2, and the recombinant Lactococcus lactis strain may be a recombinant vaccine strain transformed with a recombinant plasmid comprising the nucleotide sequence of SEQ ID NO: 1.

In one embodiment herein, the recombinant Lactococcus lactis may be a recombinant Lactococcus lactis BFE920 strain deposited under accession number KCCM11383P.

In one embodiment of the invention, the Lactococcus lactis strain BFE920 is a gram-positive, lactic acid bacterium with a generally regarded as safe (generally regarded as safe) grade lactic acid bacteria, LAB).

In one embodiment of the invention, the recombinant Lactococcus lactis strain BFE920 may be one that expresses a fusion antigen. For example, the recombinant Lactococcus lactis BFE920 strain may be a recombinant vaccine strain transformed with a recombinant plasmid, and may be selected from the group consisting of Edwards elataarum outer membrane protein A (OmpA), flagella hook protein D (FlgD), flexible glycine / serine A flexible glycine / serine linker (G / S linker), or a fusion protein of an antigen comprising all of the above.

In one embodiment of the present invention, the hairpin hook protein D may exhibit an adjuvant effect in the recombinant Lactococcus strain BFE920. For example, if the recombinant Lactococcus BFE920 strain is the Edwards City El Tatarda When expressing a fusion antigen comprising both the outer membrane protein A (OmpA), the flagellar hook protein D (FlgD), and the flexible glycine / serine linker, the single subunit strain expressing only OmpA, the wild type lactose But may not be limited to, the expression of TLR5M and / or IL - 12p40 , as compared to a strain expressing Caucus BFE920 or a control without the recombinant Lactococcus lactis BFE920 strain.

In one embodiment herein, the recombinant Lactococcus lactis strain BFE920 is selected from the group consisting of CD4-1, CD4-2, or CD8 alpha Lt; RTI ID = 0.0 &gt; mRNA &lt; / RTI &gt; expression of the gene. For example, when the recombinant Lactococcus lactis BFE920 strain is administered to fish, a control group (referred to as " Ctrl &quot; throughout the specification) or a wild-type Lactococcus lactis CD4-1 , CD4-2, or CD8? In the fish test group to which the recombinant Lactococcus lactis BFE920 was administered compared to the control group (referred to as &quot; WT LL &quot; The amount of mRNA expression relative to the gene may be increased and thus the recombinant Lactococcus lactis BFE920 strain may increase the activity of fish immunoregulatory T cells and consequently exhibit immunity enhancing activity.

In one embodiment of the present invention, the recombinant Lactococcus lactis strain BFE920 may be characterized in that intestinal permeability is reduced, but the present invention is not limited thereto. For example, the recombinant Lactococcus lactis strain BFE920 may be characterized by reducing the increased intestinal permeability upon infection with a pathogen, but may not be limited thereto.

In one embodiment herein, the pathogen can be a general hospital uniform that increases the permeability, for example Edwardsiella et al. tarda ).

The Edward Celastarda is a gram-negative bacterium having a broad range of viability, such as a temperature of about 14 ° C to about 45 ° C, a pH of about 4.0 to about pH 10.0, and a sodium chloride of about 0% to about 4% It may be a feature that is difficult to completely remove or thoroughly prevent infection because it is a sex pathogen. The Edward Celta Tarda has been found in the world through the wounds or intestines of the epithelium through water from infectious sources (e.g., feces, water, mud) under inadequate environmental conditions such as high water temperature, deteriorated water, eutrophication, It can be infected causing serious systemic bacterial disease.

In one embodiment of the invention, the recombinant Lactococcus lactis strain BFE920 may be one that increases the pathogen-specific antibody response. For example, when the recombinant Lactococcus lactis BFE920 strain was administered to fish, the recombinant Lactococcus lactis BFE920 strain was significantly inhibited from the recombinant Lactococcus lactis BFE920 strain compared with the control group to which the recombinant Lactococcus lactis BFE920 strain was not administered or the control strain to which the wild-type Lactococcus lactis BFE920 strain was administered. It may be, but not limited to, an increase in pathogen-specific antibody response in a fish test group administered with Lactis BFE920 strain.

In one embodiment of the invention, the recombinant Lactococcus lactis BFE920 strain may be one that increases the expression of Th1 subset transcription factor T-bet and / or effector cytokine IFN-y . For example, when the recombinant Lactococcus lactis BFE920 strain was administered to fish, the recombinant Lactococcus lactis BFE920 strain was significantly inhibited from the recombinant Lactococcus lactis BFE920 strain compared with the control group to which the recombinant Lactococcus lactis BFE920 strain was not administered or the control strain to which the wild-type Lactococcus lactis BFE920 strain was administered. But may not be limited to, increased expression of Th1 subset transcription factor T-bet and / or effector cytokine IFN- gamma in fish test groups treated with lactis BFE920 strain.

In one embodiment of the present invention, the recombinant Lactococcus lactis strain BFE920 may exhibit a growth promoting effect, but the present invention is not limited thereto. For example, when the recombinant Lactococcus lactis BFE920 strain was administered to fish, the recombinant Lactococcus lactis BFE920 strain was significantly inhibited from the recombinant Lactococcus lactis BFE920 strain compared with the control group to which the recombinant Lactococcus lactis BFE920 strain was not administered or the control strain to which the wild-type Lactococcus lactis BFE920 strain was administered. But may not be limited to, the increase in body weight gain and / or the decrease in feed conversion in the fish test group to which lactis BFE920 strain has been administered .

In one embodiment of the invention, the immunological enhancement may be induced in the intestine, but may not be limited thereto.

In one embodiment of the invention, the immunoenhancement may be induced by increased immune regulatory T cell activity. Specifically, the immune enhancement, but may be identified by the increase in CD4-1, CD4-2, or CD8α mRNA relative expression of the gene, it may not be limited thereto.

The immunoregulatory T cells may be classified into natrual regulatory T cells or adaptive regulatory T cells according to their origin. In the case of the normal regulatory T cells, a high level of CD25 and a cytotoxic T lymphocyte (CTLA4) or a glucocorticoid-induced TNF receptor family-related protein derived from the thymus protein, GITR), and foxp3. In the case of the adaptive regulatory T cells, it is known to secrete cytokines such as IL-10 and TGF-ss. In addition, since immunoregulatory T cells express CD4 and CD25, the immuno-promoting activity can be confirmed by increasing the relative mRNA expression of the CD4-1, CD4-2, or CD8 alpha genes.

In one embodiment of the invention, the composition may include, but is not limited to, a feed composition or a feed additive composition. For example, the composition may include a feed composition or a feed additive, and the feed composition or the feed additive composition may be used to express the anti-inflammatory or immunostimulatory activity of a fish, but the present invention is not limited thereto .

In one embodiment of the invention, the feed additive may be in the form of a high concentrate, powder, or granule, but is not limited thereto.

In one embodiment of the present invention, the feed additive may include, but is not limited to, an organic acid, a phosphate, or a natural antioxidant.

For example, the organic acid may be selected from the group consisting of citric acid, fumaric acid, adipic acid, lactic acid, malic acid, and combinations thereof, and the phosphate may be selected from the group consisting of sodium phosphate, potassium phosphate, acid pyrophosphate, And the natural antioxidant may be selected from the group consisting of polyphenols, catechins, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, But are not limited to, those selected from the group consisting of chitosan, tannic acid, phytic acid, and combinations thereof.

In one embodiment of the invention, the feed composition or feed additive may be one which contains commonly used feed ingredients. For example, the feed composition or feed additive may include, but is not limited to, cereals, vegetable protein feeds, animal protein feeds, sugars, dairy products, nutritional supplements, digestion and absorption enhancers, .

For example, the cereal may be selected from the group consisting of crushed or broken wheat, oats, barley, corn, rice, and combinations thereof, wherein the vegetable protein feed is selected from the group consisting of flour, soybean, sunflower, The animal protein feed may be one selected from the group consisting of blood, meat, bone meal, fish meal, and a combination thereof. But may not be limited.

In one embodiment of the invention, the feed additive may be administered alone to the animal, mixed directly with the animal feed as top dressing, mixed with the animal feed in a separate oral formulation separate from the feed, or mixed with an edible carrier carrier may be administered in combination with other feed additives, but the present invention is not limited thereto.

For example, when the feed additive is administered separately from an animal feed, it can be prepared in an immediate release or sustained release formulation, in combination with a pharmaceutically acceptable edible carrier as is well known in the art.

In one embodiment of the invention, the edible carrier may be a solid or a liquid, for example, the liquid may be corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol.

In one embodiment of the present invention, when the carrier is a solid, it may be, but not limited to, a tablet, a capsule, a powder, a troche or a granule or a top-dressing in a finely divided form.

In one embodiment of the invention, when the carrier is a liquid, the feed additive may be, but is not limited to, a gelatin soft capsule, or a syrup or suspension, emulsion, or solution formulation.

In one embodiment of the invention, the feed may comprise any protein-containing organic kernel meal commonly used to meet an animal's dietary needs. For example, the protein-containing flour may include, but is not limited to, corn, soy flour, or corn / soy flour mix.

In one embodiment of the present invention, the feed additive or feed composition may include, but is not limited to, an adjuvant. For example, the feed additive or feed composition may be one in which the adjuvant is additionally infiltrated, sprayed, or mixed and then used as feed for the animal or added to the feed, and may contain preservatives, stabilizers, wetting agents, emulsifiers, But the present invention is not limited thereto.

In one embodiment of the present application, the fish may be one comprising a fish that is capable of being cultured, for example, a flounder, an eel, a rockfish, a lobster, a lobster, a sea bream, a lobster, a mullet, , Horse mackerel, or ratfish, but the present invention is not limited thereto.

In one embodiment of the present application, the fish is selected from the group consisting of a moult or mortality rate at the time of infection with Edwardsiella tarda , which is significantly greater than the mortality or mortality when not infected with the Edwardsi ella tarda But it may not be limited thereto.

The second aspect of the present invention provides a pharmaceutical composition for preventing or treating Edward's disease in fish, comprising recombinant Lactococcus lactis BFE920 strain (Accession No. KCCM11383P) as an active ingredient.

Although the description of the pharmaceutical composition for the prevention or treatment of Edwards disease of fish according to the second aspect of the present application has been omitted from the description of the first aspect of the present invention, The contents can be equally applied to the second aspect of the present invention.

In one embodiment of the invention, the pharmaceutical composition for prophylaxis or therapy may further comprise a pharmaceutically acceptable carrier. Throughout this specification, the phrase " pharmaceutically acceptable " means that the composition is free of toxicity to cells or humans exposed to the composition. For example, the carrier can be a buffer, a preservative, , Isotonic agents, stabilizers, bases, excipients, lubricants, and the like, which are well known in the art.

In one embodiment of the invention, the pharmaceutical composition may be used as a formulation selected from the group consisting of oral, topical, suppository, sterile injectable solutions, skin external agents, and combinations thereof. For example, the oral form may include powders, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols, and the external preparation for skin may be an ointment, a lotion, a spray, a patch, , A suspending agent, or a gel, although the present invention is not limited thereto.

In one embodiment of the invention, the solid preparation for oral administration of the pharmaceutical composition may be a tablet, a pill, a powder, a granule, or a capsule. For example, the solid preparation may be prepared by mixing at least one excipient into the recombinant Lactococcus lactis strain BFE920, and the at least one excipient may be calcium carbonate, sucrose, or lactose ), Gelatin, magnesium stearate, or talc.

In one embodiment of the invention, the liquid formulation for oral administration of the pharmaceutical composition may be a suspension, a solution, an emulsion, or a syrup. For example, the liquid formulation may include, but is not limited to, water, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, and the like.

In one embodiment of the invention, the formulation for parenteral administration of the pharmaceutical composition may include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository, for example. Examples of the suspending agent may include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

In one embodiment of the invention, the pharmaceutical composition may comprise a carrier, excipient, or diluent for formulation. For example, the carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose , Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil, and the excipient or diluent may be a filler, an extender, a binder, a wetting agent, Releasing, or containing a surfactant, but may not be limited thereto.

In one embodiment of the invention, the pharmaceutical composition may be administered in a pharmaceutically effective amount. Throughout this specification, the term " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, But are not limited to, those factors well known in the medical arts and including factors such as health condition, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and rate of release, Lt; / RTI &gt;

In one embodiment of the invention, the pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

In one embodiment of the invention, the dosage of the pharmaceutical composition is such that the dose of the pharmaceutical composition is such that the intended use, the addiction to the disease, the age (age), weight (weight), sex, The kind, and the like. For example, the frequency of administration of the pharmaceutical composition is not particularly limited, but may be administered once a day or several times divided into doses. However, the dosages do not limit the scope of the present invention in any way, as it may vary depending on the route of administration, severity of disease, sex, weight, age and the like.

A third aspect of the present invention provides a method of preventing or treating Edward's disease in a fish, comprising the step of administering to the fish the pharmaceutical composition comprising the recombinant Lactococcus lactis strain BFE920 (Deposit No. KCCM11383P).

Although a detailed description of the method for preventing or treating Edward's disease according to the third aspect of the present application is omitted for the sake of simplicity and clarity of the description of the first aspect of the present application, The same applies to the third aspect.

1 is a flow chart illustrating a method of preventing or treating an Edwards disease according to one embodiment of the present invention. As shown in Figure 1, the fish may first be adapted in the wastewater tank for a period of about one week. The adaptation period is not limited to the entirety of the present specification, and the feed that is conventionally used may be fed during the adaptation period.

In one embodiment of the present invention, the pharmaceutical composition may contain the recombinant Lactococcus lactis strain BFE920 in which the nucleic acid molecule of SEQ ID NO: 1 is incorporated as an active ingredient.

Next, a " prime vaccination " is carried out in which the fish is administered or fed with a pharmaceutical composition containing the recombinant Lactococcus lactis BFE 920 as an active ingredient. The prime vaccination may be performed for a period of about one week, but may not be limited thereto.

In one embodiment of the present invention, in order to confirm the efficacy of the pharmaceutical composition comprising the recombinant Lactococcus lactis BFE920 as an active ingredient, the prime vaccination may be carried out using, for example, a recombinant Lactococcus lactis strain BFE920 as an active ingredient (WT LL) comprising the wild-type Lactococcus lactis BFE920 strain, a composition not containing the recombinant Lactococcus lactis BFE920 strain (Ctrl), or a single subunit comprising only one of OmpA or FlgD May be administered in divided doses.

In one embodiment of the invention, it may be one having a rest after the prime vaccination. The rest refers to a period after the prime vaccination and before the booster vaccination, which may mean a period of about one week. In the rest period, feeds that are conventionally used can be fed.

In one embodiment of the present invention, a " boost vaccination " is performed after a rest period after the prime vaccination, wherein the pharmaceutical composition comprising the recombinant Lactococcus lactis BFE920 strain as an active ingredient is administered or fed. In one embodiment herein, the detailed boost vaccination method and duration may be different from the prime vaccination.

The fourth aspect of the present invention provides a feed composition for preventing or ameliorating Edward's disease of fish, comprising recombinant Lactococcus lactis strain BFE920 (accession number: KCCM11383P) as an active ingredient.

Although the description of the feed composition for preventing or ameliorating the Edwards disease of fish according to the fourth aspect of the present invention has not been described in detail for the first and second aspects of the present application, The contents can be equally applied to the fourth aspect of the present application.

The fifth aspect of the present invention provides a feed additive composition for preventing or improving Edwards disease of fish, which comprises recombinant Lactococcus lactis strain BFE920 (accession number: KCCM11383P) as an active ingredient.

Although the description of the feed additive composition for preventing or ameliorating the Edwards disease of fish according to the fifth aspect of the present application has been omitted from the description of the first aspect of the present application, The description can be equally applied to the fifth aspect of the present application.

Herein, Ella Edward when the tar (Edwardsiella tarda ) outer membrane protein A (OmpA), flagellar hook protein D (FlgD), flexible glycine / serine linker, or both Lactobacillus lactis BFE920 ( L. lactis BFE920), wherein the recombinant Lactococcus lactis BFE920 can be used as a live feed vaccine strain for flounder ( Paralichthys olivaceus ) against Edwardsiellosis in fish.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are given to aid understanding of the present invention, and the present invention is not limited to the following examples.

[ Example ]

1. Preparation of recombinant vaccine strains

On November 20, 2014, E. tarda HFTC0081 isolated from flounder collected from Poyang Youngil fish farm was used as a template for amplification of antigen DNA. The antigen insert is a single &lt; RTI ID = 0.0 &gt; OmpA , FlgD, or the two OmpA and FlgD antigens linked by a G / S linker. In the present application, the nucleic acid molecule of SEQ ID NO: 1 contains all of the OmpA, FlgD and G / S linkers (pCYT: Fusion, Figure 2 (a) L. lactis codon for increased efficiency [SK Gupta, TK Bhattacharyya, TC Ghosh, Synonymous codon usage in Lactococcus lactis : mutational bias versus translational selection, J. Biomol. Struct. Dynam. 21 (2004) 527-536.]. PCR was performed with the primer shown in Table 1 below and the cycle shown in the cycle shown in Figure 2 (b) to amplify each antigen DNA fragment.

To form a fusion antigen insert, overlap extension PCR (OE-PCR) was performed.

In summary, the purified 5'- OmpA- G / S linker-3 'and 5'-G / S linker- FlgD- 3' PCR products were mixed at a 1: 1 molar ratio and adjusted to about 80 ng, PCR Master Mix (Takara Bio Inc., Shiga, Japan). In the absence of the primer, a denaturation step at 95 ° C for 5 minutes was performed, and an overlap anneal and extension step was performed at 70 ° C for 10 minutes. After the overlap step, OmpA-F and FlgD-R primers were added to the reaction and the remaining PCR cycles were performed as shown in Figure 2 (b) on the right (overlap extension elongation PCR cycle). Each DNA insert was ligated to pCYT using T4 DNA ligase (NEB Biolabs, Ipswich, Mass., USA) according to the manufacturer's instructions. The product had attached Gene Pulser II Electroporation System (Bio- Rad, Hercules, CA, USA) for Escherichia by electroporation using It was transformed with coli DH5 α. Positive transformants were selected from Luria-Bertani (LB, Difco Laboratories, Detroit, MI, USA) agar plates containing 25 μg / ml of flamephenycol and confirmed by PCR using pCYT vector-specific primers Table 1). Like the plasmid information shown in Figure 2 (c), the plasmid was extracted from the positive transformants and transformed with L. lactis BFE920. Vector positive L. lactis BFE920 transformants were screened on agar plates of de Man Rogosa Sharpe (MRS, Difco Laboratories, Detroit, MI, USA) containing 25 μg / ml of chloramphenicol.

Selected positive clones were further analyzed by western blot analysis to examine antigen expression. Briefly, pCYT: OmpA containing recombinant L. lactis BFE920 (rLL-OmpA), pCYT: FlgD (rLL-FlgD), or pCYT: Fusion (rLL-Fusion) clones were inoculated at 1% v / v with fresh MRS broth supplemented with 25 μg / ml chloramphenicol in starter culture and incubated overnight at 30 ° C with 200 rpm agitation. 10 ng / ml of nisin (Sigma-Aldrich, St. Louis, Mo., USA) was added to replicate samples to determine if there was an additional effect on antigen expression by the recombinant L. lactis BFE920 strain . After induction of nisin, all cultures were incubated for 2 hours at 30 DEG C with stirring at 200 rpm. The fully cultured cell pellet was collected by centrifugation at 4,500 rpm, 10 min, 4 ° C and washed with 1X PBS (1.47 mM KH ₂ PO ₄ , 4.3 mM Na ₂ HPO ₄ , 2.7 mM KCl, 137 mM NaCl, pH 7.3) Washed twice. The cell pellet was 20 fold concentrated by resuspension in urea denaturation buffer (8 M urea dissolved in 1X PBS) and bead beated using silica zirconia beads. The final supernatant was collected by centrifugation at 13,000 rpm, 10 min, 4 ° C and used for western blot analysis.

Briefly, the protein-transfected PVDF membrane was diluted 1: 1,000 with anti-6XHis Tag antibody (Biolegend, San Diego, CA, USA) as primary antibody and then blocked with 5% v / v skim milk blocking and 1: 3000 diluted HRP A conjugated goat anti-mouse secondary antibody (Bioss Inc, Woburn, MA, USA) was treated as a detection antibody. Imaging of the membranes was performed 5 times at 15 minute intervals with SuperSignal Femto Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and Omega LumG imager (Aplegen, San Francisco, CA, USA).

Through the Western blot analysis, the expression of the designated antigen was confirmed by all of the recombinant vaccine strains (FIG. 3). The OmpA, FlgD, and fusion were successfully expressed in each recombinant L. lactis BFE920 strain at 10 ng / mL. Proteins expressing the fusion antigen appeared at 62 kDa, 37 kDa at OmpA and 24 kDa at FlgD, depending on the protein size marker. Each antigen was expressed without nisin induction. Since the assay was performed in an semi-quantitative manner, there was no difference in expression levels compared to additional nisin treatment.

2. Experimental animal and feed vaccination

A healthy flounder ( Paralichthys olivaceus ) weighing 86.36 ± 4.31 g purchased from Pohang's Friendship Hatchery and Farm was adapted at 20-21 ° C for 7 days in a 2,000 L cylindrical aquarium. Five fish per experimental group were placed in a wastewater tank as follows: Ctrl, conventional food; WT LL, wild type L. lactis BFE920; OmpA, rLL-OmpA payroll; FlgD, rLL-FlgD pay; Fusion, rLL-Fusion salary. An additional 20 fish were also prepared to investigate the recall function of the best vaccine based on the vaccination results. Each bacterial strain was incubated overnight (~ 1.4 × 10 ⁹ CFU / mL) and was used as reference [D. Kim, BR Beck, SM Lee, J. Jeon, DW Lee, JI Lee, et al., Pellet feed adsorbed with the recombinant Lactococcus lactis BFE920 expressing SiMA antigen against strong recall vaccine against Streptococcus iniae infection in olive flounder ( Paralichthys olivaceus ), Fish Shellfish Immunol. About 0.8 × 10 ⁷ CFU / g to 1.4 × 10 ⁷ CFU / g in a commercial extruded pellet feed (Flounder Bluechip; Woosugn Co., Daejeon, Korea) Was mixed with the final bacterial concentration. Priming and boosting feed vaccines were performed for 7 days per inoculation. During rest days, fish were fed commercially available feed. The fish were fed vaccines or commercially available feed twice daily at 9:00 and 18:00. The entire vaccination process is shown in FIG.

3. Sample collection, RNA extraction, and cDNA synthesis

On day 7 of vaccination, intestinal tissue was collected from 5 fish per group and immediately placed in 800 μL of RNAlater® solution (Ambion, Austin, TX, USA). After stabilization at 4 ° C overnight, the samples were stored at -80 ° C until RNA extraction proceeded. The 40 mg-weight intestinal tissue was resuspended in lysis buffer (TaKaRa Mini Best Universal RNA Extraction Kit, Takara Bio Inc., Tokyo, Japan) using 600 μL of T10 basic handheld homogenizer (IKA®, China) Shiga, Japan). RNA quality and quantity were measured using SPECTRO STANNO (BMG LABTECH, Ortenberg, Germany). A total of 5 μg of cDNA per sample was then generated using SuperScript® III reverse transcriptase (Invitrogen, Carlsbad, USA) and random hexamer primers according to the manufacturer's instructions. Blood samples (approximately 800 μL) were collected in heparinized tubes, such as intestinal tissue sampled through the fish's tail vein. Plasma samples were collected by centrifugation at 13,000 rpm, 10 min, 4 ° C.

After the feed vaccination, the T-cell response of the flounder was determined in the intestine of each experimental group by CD4-1, CD4-2, CD8 &lt; / RTI &gt; gene expression levels. There was no significant difference between the vaccine groups after the prime vaccination, but all vaccine groups had higher T cell marker gene expression than the Ctrl and WT LL groups (Fig. 4 (a) - Fig. 4 (c)). After the boost vaccination, all vaccine groups showed an increased T cell response compared to the Ctrl group (Fig. 4 (d) to Fig. 4 (f)). In addition, Fusion group significantly increased CD4-2 and CD8 alpha Gene expression. There was no statistical significance between CD4-1 expression in the Fusion group and the FlgD group, but the expression level was higher in the Fusion group.

In addition, expression of Th1 subset transcription factors T-bet and IFN- y in the experimental group was compared to confirm the inflammatory response vaccine effect. The expression of T-bet was significantly higher in the vaccine group than in the WT LL group, and only the Fusion group showed statistical significance after the prime vaccination compared to the control group (Fig. 5 (a)). IFN- y expression levels were increased after the first inoculation in the WT LL group and the vaccine group compared to the control group (Fig. 5 (b)). After the boost vaccination, T-bet and IFN- y expression was significantly increased in all the vaccine groups compared to the Ctrl group, and the Fusion group showed the highest expression of the Th1 subset gene compared to the single antigen vaccine (c) and (d)].

In order to investigate the innate immune response of the flounder to the vaccine strain, gene expression levels of TLR5M , IL- 1 [beta] , and IL- 12p40 were measured in the field of each experimental group. Expression of TLR5M was significantly increased in the rLL-FlgD or rLL-Fusion group compared to the other groups (Fig. 6 (a)). This effect was not observed in the OmpA group. However, IL- 1β was increased in all vaccine groups, and there was no statistical difference between the vaccine groups (FIG. 6 (b)). Interestingly, IL- 12p40 in the FlgD and Fusion groups The response was significantly increased, which is associated with an increased TLR5M response (Fig. 6 (c)). Expression levels of IL- 12p40 in OmpA and WT LL were lower than those in the FlgD and Fusion groups, but higher than those in the Ctrl group (OmpA, p <0.001; WT LL, p = 0.20) The reason is due to the immunostimulatory effect of L. lactis BFE920 itself.

4. Serum E. tarda  Specific antibody response

The E. tarda- specific antibody response was [D. Kim, BR Beck, SM Lee, J. Jeon, DW Lee, JI Lee, et al., Pellet feed adsorbed with the recombinant Lactococcus lactis BFE920 expressing SiMA antigen against Streptococcus iniae infection in olive flounder ( Paralichthys olivaceus ) , Fish Shellfish Immunol. 55 (2016) 374-383. &Lt; / RTI &gt;

In summary, the antigen was prepared by sonication of overnight cultured E. tarda HFTC0081 resuspended in Na ₂ CO ₃ / NaHCO ₃ pH 9.6 coated buffer. Antigen solution was dispensed into each well of a 96 well plate (Corning Inc., Kennebunk, ME, USA) and incubated overnight at 4 ° C. After coating with the antigen, the mouse-anti-flounder antibody diluted with 1: 1,000 diluted with 1: 800 as the primary antibody and the diluted 1: 1,000 as the secondary antibody, and the tertiary / detection Mouse secondary antibody (Bioss Inc, Woburn, MA, USA) diluted 1: 4,000 as the antibody. To measure the colorimetric absorbance, 100 μL of a 3,30,5,50-tetramethylbenzidine solution (Calbiochem, Darmstadt, Germany) was treated in each reaction well and the reaction was terminated by treatment with 2 NH ₂ SO ₄ . The absorbance of the reaction was measured at 450 nm using SPECTROstar Nano (BMG LABTECH, Ortenberg, Germany).

E. tarda- specific antibody levels in the sera of the experimental group were confirmed during each vaccination schedule as a B cell response test. Increased E. tarda specific antibody levels were detected in both the prime vaccine (Figure 7 (a)) and the boost vaccination (Figure 7 (b)) compared to the Ctrl and WT LL groups of all vaccine groups. There was no significant difference between the vaccine groups at each vaccination. The absorbance level was increased in the above vaccination vaccine of all experimental groups due to fish growth for 2 weeks.

5. qRT-PCR (Quantitative real-time PCR) analysis

QRT-PCR analysis of CD4-1, CD4-2, CD8α, T- bet, IFN -γ, IL- 1β, IL- 12p40, and TLR5M this was carried out to measure the effect of the vaccine halibut. The primers for the qRT-PCR reaction in the above analysis are shown in Table 2.

To summarize, 100 ng of cDNA samples obtained from the field of each fish were amplified using SYBR® Premix Ex Taq ^™ (Applied Biosystems 7500 PCR System, Applied Biosystems, Foster City, CA, USA) (Tli RNaseH plus) (Takara Bio Inc., Shiga, Japan) in a volume of 20 μL per PCR reaction. The qRT-PCR cycle is as follows: denaturation at 94 ° C for 15 seconds after the first denaturation at 94 ° C for 10 minutes, and 40 cycles of annealing / extension for 45 seconds at 62 ° C. Melting curves were analyzed using a programmed melt-curve cycle of a 7500 PCR system and the relative expression of each gene was determined using the 2 ^- ΔΔΔCT method [KJ Livak, TD Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR ^{and the 2 -. △△ CT Method} , Methods 25 (2001) 402-408] was calculated with the β-actin as a control, using ΔCT.

6. E. tarda HFTC0081  Inoculation experiment

During the last resting week of the feed vaccination schedule, each of the 40 fish per group was administered intraperitoneally to 5 × 10 ⁶ CFU E. tarda HFTC0081 per fish, which showed approximately LD80 dose within 7 days after dosing (Table 3).

In order to clearly compare the protective efficacy among the rLL-OmpA, rLL-FlgD, or rLL-Fusion feed vaccinated groups, doses higher than LD50 were inoculated. The relative survival rate (RPS) per group was calculated by the following equation.

At 14 days after the inoculation, CT survived 10.0% of survival rates for E. tarda infection, 37.5% for WT LL, 50.0% for OmpA, 55.0% for FlgD, and 82.5% for Fusion group (Fig. 8). All vaccine and WT LL groups showed statistical significance compared to the control group. However, there was no significant difference between WT LL, OmpA and FlgD groups. The survival rate of the Fusion group was significantly higher than that of all other experimental groups. The transformed RPS values of each experimental group were as follows: WT LL, 30.6%; OmpA, 44.4%; FlgD, 50.0%; Fusion group, 80.6%.

7. Large-scale vaccination

In order to confirm that the feed vaccines and protocols provided herein can be applied in actual farms, large-scale feed-inoculations have been carried out in Pohang, Korea. The flounder, weighing 131.96 ± 4.50 g, was placed in a 10m × 10m water bath. The experimental groups were set up as follows: 1,500 fish per aquarium, 2 control tanks, and 2 tanks vaccine groups, totaling 6,000. The rLL-Fusion strain was selected for field application depending on the vaccine efficacy outcome. Natural mortality and feed intake were monitored during the 4-week vaccination schedule. Percent weight gain (PWG) and feed conversion ratio (FCR) were calculated as follows:

PWG = 100 x {final weight (g) - initial weight (g)} / initial weight (g)

FCR = feed consumption (g) / {final weight (g) - initial weight (g)}

(FIG. 9) during the in-field challenge inoculation (Figure 9), with increased weight gain (PWG: control, 39.89%; vaccine group, 61.83%) and reduced feed conversion (FCR: control; 2.34; Six measurement methods for 20 randomly selected fish were presented with standard error (SEM). In addition, no significant natural history was observed in both experimental groups (4-week cumulative mortality: 0.40% in the control, 0.33% in the vaccine group).

8. Statistical analysis

The qRT-PCR and antigen-specific antibody ELISA results were analyzed using two-tailed Student t-test with Turkey's multiple comparison post-test using Graph Pad Prism 5 software. Were analyzed by unpaired, two-tailed student's t-test and one-way analysis of variance (ANOVA). The significance of survival curves after E. tarda HFTC0081 inoculation was analyzed by log-rank (Mantel-Cox) test using Graph Pad Prism 5 software.

T cell activation during vaccination is important because it induces adaptive immune and memory T cells in response to the processing antigen expressed by antigen presenting cells [SM Kaech, EJ Wherry, R. Ahmed, Effector and memory T-cell differentiation : implications for vaccine development, Nat. Rev. Immunol. 2 (2002) 251-262.], [RA Seder, PA Darrah, M. Roederer, T-cell quality in memory and protection: implications for vaccine design, Nat. Rev. Immunol. 8 (2008) 247-258.], [D. Masopust, JM Schenkel, The integration of T cell migration, differentiation and function, Nat. Rev. Immunol. 13 (2013) 309-320.]. It increased after vaccination CD4-1, CD4-2, or transcription levels of CD8α means the proper functioning of the recombinant vaccine strain to the oral treatment of the present application. In addition, the Thl subset is important during vaccination due to the proinflammatory effector function of activating other immune cells [SL Swain, KK McKinstry, TM Strutt, Expanding roles for CD4 + T cells in immunity to viruses, Nat . Rev. Immunol. 12 (2012) 136-148.]. Th1 subset activation by increased T-bet with significantly increased IFN- y transcription compared to the control supports that the vaccine strain used herein successfully stimulated T cell immunity. In addition, the adjuvant effect of recombinant L. lactis BFE920 was further confirmed here as the levels of IL-1β and IL-12p40 expression were increased in the wild-type L. lactis BFE920 and vaccine-fed groups. These results confirm that the adjuvant effect of recombinant L. lactis BFE920 can overcome oral tolerance when a particular recombinant antigen is expressed therefrom.

Interestingly, the fusion vaccine feed group (Fusion) showed increased T cell response compared to the single subunit vaccine feed group (OmpA group or FlgD group). Similar elevated vaccine effects of multiple subunit antigens as compared to single subunit antigens have also been demonstrated in the tuberculosis model [A. Olsen, L.A.H. van Pinxteren, L.M. Okkels, P.B. Rasmussen, P. Andersen, Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85B and ESAT-6, Infect. Immun. 69 (2001) 2773-2778., J.A.M. Langermans, T.M. Doherty, R.A.W. Vaccine 23, (2005) 2740-8, 1999. Vaccine, T. van der Laan, K. Lyashchenko, R. Greenwald et al., Protection of macaques against Mycobacterium tuberculosis infection by a subunit vaccine based on a fusion protein of antigen 85B and ESAT- 2750.] and Chlamydia infection model [AW Olsen, L.A.H. van Pinxteren, L.M. Okkels, P.B. Rasmussen, P. Andersen, Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85B and ESAT-6, Infect. Immun. 69 (2001) 2773-2778., [A. Olsen, M. Theisen, D. Christensen, F. Follmann, P. Andersen, Protection against Chlamydia promoted by a subunit vaccine (CTH1) compared with a mouse genital challenge model, PLoS ONE 5 (2010) e10768. doi: 10.1371 / journal.pone.0010768.]. Also, Eko et al., 2004 [F.O. Eko, Q. He, T. Brown, L. McMillan, G.O. Ifere, G.A. Ananaba et al., A novel recombinant multisubunit vaccine against Chlamydia, J. Immunol. 173 (2004) 3375-3382.) Showed that it is possible to perform Th1 subset stimulation using fusion antigens. The exact mechanism is unclear, but as the number of epitopes that can be sensitized to immune cells increases, a more diverse antigen presentation repertoire can be provided.

In addition to the abundance and diversity of epitopes, binding of effective antigens is another key to improving vaccine effectiveness. From the innate immunity point of view, the expression of TLR5M and IL-12p40 in the rLL-FlgD and rLL-Fusion groups was significantly increased compared to rLL-OmpA suggesting that the FlgD subunit stimulated innate immunity. In particular, TLR5 (Toll-like receptor 5) is a pattern recognition receptor in which the ligand is a bacterial flagellate, and when activated, promotes an inflammation-promoting cascade through the adapter protein Myd88 and the transcription factor NF-kB [F. Hayashi, K.D. Smith, A. Ozinsky, T.R. Hawn, E.C. Yi, D.R. Goodlett, The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5, Nature 410 (2001) 1099-1103. Akira, K. Takeda, Toll-like receptor signaling, Nat. Rev. Immunol. 4 (2004) 499-511.], [L.A.J. O'Neill, D. Golenbock, A.G. Bowie, The history of Toll-like receptors- redefining innate immunity, Nat. Rev. Immunol. 13 (2013) 453-460.]. Although there is no direct evidence of FlgD as a TLR5 ligand, the TLR5-stimulating activity of FlgD is presented here by increased TLR5M expression. In addition, the flagella flagella E (FlgE) of Pseudomonas aeruginosa has a proinflammatory effect [Y. Wang, Y. Shen, Q. Yuan, D. Lin, M. Wang, Z. Liang et al., Flagellar hook protein FlgE initiates a proinflammatory response via mixed pathways (MPF2P. J. Immunol. 194 (1 Supplement) (2015) 63.21.]. The flagella subunit, FliC [M.R.A. Vaccine 31 (2013) 1210-1216.], [G (G), M. Oloomi, M. Mahdavi, M. Habibi, S. Bouzari, Vaccination with recombinant FimH fused with flagellin enhances cellular and humoral immunity against urinary tract infection in mice, . Yin, M. Qin, X. Liu, J. Suo, X. Tang, G. Tao, et al., An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin, Biochem . Biophys. Res. Commun. 440 (2013) 437-442.] Or FlaB [C. Nguyen, S.Y. Kim, M.S. Kim, S.E. Lee, J.H. Other studies on Rhee, Intranasal immunization with recombinant PspA fused with a flagellin enhances cross-protective immunity against Streptococcus pneumoniae infection in mice, Vaccine 34 (2011) 5731-5739.], As well as with recombinant vaccine antigen design as an effective fusion partner We have proposed this strategy. Thus, our researchers suggest that E. tarda FlgD is a useful adjuvant that can be used simultaneously as antigen and adjuvant.

Flagella [SB Mizel, JT Bates, Flagellin as an adjuvant: cellular mechanisms and potential, J. Immunol. 185 (2010) 5677-5682.], A complementary subunit [ES Bergmann-Leitner, WW Leitner, GC Tsokos, Complement 3d: From molecular adjuvant to immune escape mechanisms, Clin. Immunol. Protein-based adjuvants such as the non-peptides &lt; RTI ID = 0.0 &gt; (A) &lt; / RTI &gt; It has two major advantages over adjuvants: 1) it can be co-expressed by a vaccine vehicle, 2) it can be bound directly to the desired antigen using a peptide linker. However, in order to produce an effective fusion antigen, it is preferred that a flexible or rigid peptide linker is capable of affecting antigen safety, protein folding, biological activity and / or antigen expression levels in the transformant, Should be considered [X. Chen, JL Zaro, W.-C. Shen, Fusion protein linkers: Property, design and functionality, Adv. Drug Deliv. Rev. 65 (2013) 1357-1369.]. E. tarda protection was better in rLL-Fusion treated group than in other vaccine groups, but there was no difference in vaccine-specific antibody levels between the groups. This phenomenon can be regarded as a significant enhancement of T cell and innate immune activities by fusion antigens because T cell activity has a crucial effect on intracellular pathogen infection [SHE Kaufmann, Immunity to intracellular bacteria, Annu. Rev. Immunol. 11 (1993) 129-163.]. E. tarda can survive and avoid the host's immune system through survival and replication in phagocytosis and epithelial cell invasion [SB Park, T. Aoki, and TS Jung, Edwardsiella tarda infection in fish, Vet. Res. 43 (2012) 67.]. Thus, improved T cells and innate immunity can contribute to the survival of flounder with effective protection against intracellular E. tarda.

The probiotic and immunostimulatory effects of the recombinant L. lactis BFE920 were determined by the flounder model [D. Kim, BR Beck, S.-B. Heo, J. Kim, HD Kim, S.-M. Lee et al., Lactococcus lactis BFE920 activates the innate immune system of olive flounder ( Paralichthys olivaceus ), resulting in protection against Streptococcus iniae infection and enhancing feed efficiency and weight gain in large scale field studies, Fish Shellfish Immunol. 35 (2013) 1585-1590.], [BR Beck, D. Kim, J. Jeon, S.-M. Lee, HK Kim, O.-J. Kim, et al., The effects of combined dietary probiotics Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 on innate immunity and disease resistance in olive flounder ( Paralichthys olivaceus ), Fish Shellfish Immunol. 42 (2015) 177-183.], [BR Beck, JH Song, BS Park, D. Kim, J.-H. Kwak, HK Do, et al., Distinct immune tones are established by Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 in the gut of olive flounder ( Paralichthys olivaceus ), Fish Shellfish Immunol. 55 (2016) 434-443.] And the advantages of L. lactis species as a live vaccine, Casalta and Montel [E. Casalta, M.-C. Montel, Safety assessment of dairy microorganisms: The Lactococcus genus, Int. J. Food Microbiol. 126 (2008) 271-273. In view of the vaccine delivery system , L. lactis BFE920 does not colonize the intestine, so [N. Mielcarek, S. Alonso, C. Locht, Nasal vaccination using live bacterial vectors, Advan. Drug Del. Rev. (2001) 55-69.], The risk of oral tolerance development due to prolonged antigen exposure is low. Another advantage of recombinant L. lactis BFE920 as an antigen producing microbial device is nisin production [CMAP Franz, M. Du Toit, A. von Holy, U. Schillinger, WH Holzapfel, Production of nisin-like bacteriocins by Lactococcus lactis strains isolated from vegetables, J. Basic Microbiol. 37 (1997) 187-196.]. Although an inducible nisin promoter (PnisA) is used in the plasmid, the antigen is continuously expressed and self-induced by the nisin produced by the vehicle.

In addition to the advantages of recombinant L. lactis BFE920 as a vaccine vehicle, there is interesting but conflicting results from the L. lactis BFE920 study in terms of immunology. IFN- ? Stimulation by L. lactis BFE920 has been reported several times [D. Kim, BR Beck, S.-B. Heo, J. Kim, HD Kim, S.-M. Lee et al., Lactococcus lactis BFE920 activates the innate immune system of olive flounder ( Paralichthys olivaceus ), resulting in protection against Streptococcus iniae infection and enhancing feed efficiency and weight gain in large-scale field studies, Fish Shellfish Immunol. 35 (2013) 1585-1590.], [BR Beck, JH Song, BS Park, D. Kim, J.-H. Kwak, HK Do, et al., Distinct immune tones are established by Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 in the gut of olive flounder ( Paralichthys olivaceus ), Fish Shellfish Immunol. 55 (2016) 434-443.], [D. Kim, BR Beck, SM Lee, J. Jeon, DW Lee, JI Lee, et al., Pellet feed adsorbed with the recombinant Lactococcus lactis BFE920 expressing SiMA antigen against strong recall vaccine against Streptococcus iniae infection in olive flounder ( Paralichthys olivaceus ), Fish Shellfish Immunol. (Figure 10) [BR Beck, JH Song, BS Park &lt; RTI ID = 0.0 &gt; , D. Kim, J.-H. Kwak, HK Do, et al., Distinct immune tones are established by Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 in the gut of olive flounder ( Paralichthys olivaceus ), Fish Shellfish Immunol. 55 (2016) 434-443.]. Nonetheless, the vaccine effect was well documented in S. inae and E. tarda models. Regarding this inconsistency, there are several reports that a regulatory T cell (Treg) subset is important in a particular vaccination process. In one study, CD8 + T cell infiltration was stimulated by Treg cells [TJ Ruckwardt, KL Bonaparte, MC Nason, BS Graham, and Regulatory T cells promoting early influx of CD8 + T cells in the lungs of respiratory syncytial virus-infected mice and diminish immunodominance disparities, J. Virol. 83 (2009) 3019-3028.], As well as the promotion of Th17 subset by Treg cells [Y. Chen, CJ Haines, I. Gutcher, K. Hochweller, WM Blumenschein, T. McClanahan, et al., Foxp3 + Regulatory T Cells Promoting Helper 17 Cell Development In Vivo through Regulation of Interleukin-2. Development of Resident Memory T Cells [JB Graham, A. Da Costa, JM Lund, Regulatory T cells shape resident memory T cell response to virus infection in the tissues, J. Immunol. 192 (2014) 683-690.] And the important role of the Treg subset for antigen priming of CD4 + T cells upon infection [AG Soerens, A. Da Costa, JM Lund, priming upon mucosal infection, Mucosal Immunol. 9 (2016) 1395-1406.] Has also been published. The mechanism of Tregs participating in mucosal vaccination is not yet clear, but it will be a topic to be solved in future studies.

Here, the rLL-Fusion vaccine strain developed demonstrates significantly improved T cell and Thl subset responses, adjuvant effects and protection against Edwards disease compared to the rLL-OmpA and rLL-FlgD vaccines expressing a single antigen.

During prime and boost vaccination, increased expression of CD4-1, CD4-2, and CD8α was observed in the vaccine fed group compared to wild type L. lactis BFE920 (WT LL) or control (Ctrl) . In addition, Th1 subset transcription factor T-bet and effector cytokine IFN- y expression was increased in the vaccinated group compared to WT LL or Ctrl. In addition, the expression levels of T cells and Thl subset genes were significantly lower than those of other groups (rLL-OmpA and rLL-FlgD) treated with a single subunit expressing a single vaccine strain during boost vaccination, Was significantly higher in the flounder fed with the recombinant L. lactis BFE920. In addition to T cell immunity, pathogen - specific antibody responses were increased in the feed vaccinated group in both prime and boost vaccination compared to WT LL or Ctrl.

From the innate immunity point of view, the flounder fed rLL-FlgD or rLL-Fusion showed an increased expression of TLR5M and IL- 12p40 , exhibiting the adjuvant effect of FlgD over rLL-OmpA, WT LL, or Ctrl. The rLL-Fusion-treated flounder was compared with the other experimental groups for E. tarda inoculation (Ctrl, 10.0%; WT LL-fed, 37.5%; rLL-OmpA-fed, 50.0%; rLL-FlgD-fed, 55.0% The highest survival rate was 82.5%. In large scale feeding experiments, the vaccine group showed increased weight gain and reduced feed conversion compared to the control group, confirming the growth promoting effect of the vehicle, which is recombinant L. lactis BFE920.

In addition, since the safety of the vehicle and the probiotic effect are assured, the vaccine system including the vaccine composition for immunization according to the present invention can be said to be more developed than the conventional vaccines used in aquaculture.

In conclusion, the rLL-Fusion vaccine strain of the present invention is a promising vaccine candidate for E. tarda infection used in aquaculture in view of the T cell immunity, antibody response, superior adjuvant effect, and protection result of the present invention.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Korea Microorganism Conservation Center (overseas) KCCM11383P 20130221

Claims (15)

A vaccine composition for immunization comprising the nucleic acid molecule of SEQ ID NO: 1 as an active ingredient.
The method according to claim 1,
SEQ ID NO: 1 is a recombinant Lactococcus lactis lactis ). &lt; / RTI &gt;
The method according to claim 1,
Wherein said SEQ ID NO: 1 encodes SEQ ID NO: 2.
3. The method of claim 2,
Wherein the recombinant Lactococcus lactis strain is a recombinant Lactococcus lactis BFE920 strain deposited with Accession No. KCCM11383P.
3. The method of claim 2,
Wherein the recombinant Lactococcus lactis strain BFE920 increases mRNA expression of the CD4-1, CD4-2, or CD8? Gene.
3. The method of claim 2,
Wherein said recombinant Lactococcus lactis strain BFE920 increases the pathogen-specific antibody response.
3. The method of claim 2,
Wherein said recombinant Lactococcus lactis strain BFE920 increases the expression of Th1 subset transcription factor T-bet .
3. The method of claim 2,
Wherein said recombinant Lactococcus lactis strain BFE920 increases the expression of effector cytokine IFN-y .
The method according to claim 6,
The pathogen is Edwardsiella &lt; RTI ID = 0.0 &gt; tarda ). &lt; / RTI &gt;
The method according to claim 1,
Wherein said immune enhancement is induced by increased immune regulatory T cell activity.
The method according to claim 1,
Wherein the immunological enhancement is induced in the intestines.
A pharmaceutical composition for preventing or treating Edward's disease in fish, which comprises as an active ingredient a recombinant Lactococcus lactis BFE920 strain (SEQ ID NO: KCCM11383P) in which the nucleic acid molecule of SEQ ID NO: 1 is incorporated.
A method for preventing or treating Edward's disease in a fish, comprising administering to the fish a pharmaceutical composition for the prevention or treatment of Edwards disease of a fish according to claim 12.
A feed composition for preventing or ameliorating the Edwards disease of fish, comprising as an active ingredient a recombinant Lactococcus lactis BFE920 strain (SEQ ID NO: KCCM11383P) in which a nucleic acid molecule of SEQ ID NO: 1 is incorporated.
A feed additive composition for preventing or ameliorating Edwards disease of fish, comprising as an active ingredient a recombinant Lactococcus lactis BFE920 strain (accession number: KCCM11383P) containing a nucleic acid molecule of SEQ ID NO: 1 as an active ingredient.

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