WO2018124407A1

WO2018124407A1 - Vaccine composition containing attenuated salmonella mutant as active ingredient for simultaneously preventing or treating porcine proliferative enteropathy and salmonella

Info

Publication number: WO2018124407A1
Application number: PCT/KR2017/006150
Authority: WO
Inventors: 이존화; 원가연; 김제형; 박수연; 문성철; 정호경
Original assignee: 주식회사 코미팜
Priority date: 2016-01-04
Filing date: 2017-06-13
Publication date: 2018-07-05
Also published as: KR101893827B1; KR20170081569A

Abstract

The present invention relates to a vaccine composition containing an attenuated Salmonella mutant as an active ingredient for simultaneously preventing or treating porcine proliferative enteropathy and Salmonella. The present invention was confirmed to induce humoral and cell-mediated immune responses to antigens in a mouse vaccinated with a mixture of the attenuated Salmonella mutant, when the attenuated salmonella was transformed using a vector developed to increase secretion of Lawsonia intracellularis-derived OptA, Optb, FliC or Hly antigens. Therefore, the mutant according to the present invention is expected to be useful as a vaccine for simultaneously preventing or treating porcine proliferative enteropathy and Salmonella, which is safe, economical, and safely inoculated.

Description

Vaccine composition for simultaneous prevention or treatment of pig proliferative ileitis and Salmonellosis comprising attenuated Salmonella mutants

The present invention relates to a vaccine composition for the simultaneous prevention or treatment of swine proliferative ileitis and salmonellosis comprising attenuated Salmonella mutant strain as an active ingredient.

Porcine proliferative enteritis (PPE) is a disease caused by Lawsonia intracellularis , which is widespread all over the world, causing significant damage to pig farmers. The incidence rate is known to be around 30% worldwide, and in 2000, the US National Animal Health Monitoring System (NAHMS) survey reported that more than one-third of all farms and 75% of large farms were infected. In Korea, more than 53% of the positive rates of individual pigs surveyed were reported.

PPE-based diseases caused by thickening of the ileum wall due to proliferation of enterocytes can be divided into proliferative hemorrhagic enteropathy and chronic incineinal adenomatosis, acute type of pig hemorrhagic intestine. The disease develops in recently stocked pigs or pigs, with red and black tars around the anus before mortality, weakness, anemia, and anorexia. Chronic type has slower feed intake, weight loss, and major clinical symptoms are brown, watery diarrhea that lasts from several days to four weeks. Statistics show that during the PPE epidemic, daily weight gains are reduced by 9-35% and feed efficiency is 6-20%, causing significant economic losses to farmers.

Compared to the prevalence and severity of farms, Rossonia Intracellularis, an obligate intracellular bacterium, is difficult to culture in vitro and has a wide variation in its growth rate. As a result, the limitations of the diagnostic analysis of this disease have limited the development of therapeutic and diagnostic methods as well as detailed pathogenesis. Conventional PCR and serological diagnostic methods are used to diagnose clinical lesions, but their sensitivity is low in the diagnosis of suspected disease or suspected host species. Because of this, it is difficult for livestock farmers to find out whether the disease is occurring on their own farms due to the nature of the disease, which is not obvious in diarrhea. Antibiotics susceptible to intracellular proliferation Gram-negative bacteria, such as tyrosin, tetratracyclines, and lyomycin, have been used to prevent the treatment of this disease, but have been practiced in Korea since July 2011. Prohibition of antibiotics in feeds has increased the need to develop effective vaccines to control the bacteria present on pig farms.

Pigs with diarrhea may not be able to pinpoint the cause of the disease based solely on clinical symptoms or diarrhea. In particular, diarrhea caused by fibrous necrotic ulcerative colitis caused by swine salmonellosis, which is a representative swine diarrheal disease, is mainly caused by Salmonella typhimurium . Salmonella, a Gram-negative bacterium that is characterized by intracellular proliferation, such as Lawsonia , has been shown to have the same clinical symptoms as well as a significant correlation with Salsonella in pigs with Salmonella. The study found that due to the action of the intestinal microflora in pigs, pigs infected with Rossonia bacteria increased the colonization of Salmonella and improved shedding. The interaction between these two bacteria suggests an urgent need for the development of a vaccine to prevent both diseases at the same time.

Significant decreases in B cells involved in humoral immunity, the number of T cells involved in cellular immunity, and the signaling lymphocytic activation molecule (SLAM7), the receptor for expression of T cells, were observed in pigs heavily infected with Lawsonia intracellularis. . Reduction of total lymphocyte counts and limited activation of cytotoxic T cells at these infected sites is known to be related to the composition of the environment and hence the regulation of immune responses that are suitable for survival of Lawsonia intracellularis. There is a need for the development of a vaccine having high antigenicity and stability to overcome the immunosuppressive response that may occur in the development of such PPE.

Meanwhile, Korean Patent No. 0758614 discloses 'Cultivation of Lausonia Intracellulose, Anti-Lausonia Intracellular Vaccine and Diagnostic Reagent', and Korean Patent No. 1151004 discloses that the adhesion factor of bovine pathogenic E. coli Transformed attenuated Salmonella mutants and vaccine compositions for preventing and treating Escherichia coli and Salmonella bacterium comprising the same are disclosed, but pig proliferative ileitis comprising the attenuated Salmonella mutants of the present invention as an active ingredient and There is no description of vaccine compositions for simultaneous prophylaxis or treatment of Salmonellosis.

The present invention is derived from the above requirements, the inventors of the present invention to enhance the immune response due to the vaccine and to prevent two types of swine diarrhea at the same time four antigens derived from Lawsonia Intracellulose (OptA, OptB, FliC, Hly) was cloned into a recombinant vector for a live vaccine containing a Bla signal sequence to transform the LPS O-antigen-deficient attenuated Salmonella strain and inoculated with the humoral and cellular antigens in the mice inoculated with the transformed Salmonella mixture. By confirming that a mediated immune response is induced, the present invention has been completed.

In order to solve the above problems, the present invention provides attenuated Salmonella mutants comprising any one antigen selected from the group consisting of OptA, OptB, FliC and Hly antigen derived from Lawsonia intracellularis .

In addition, the present invention is Lawsonia Intracellulose ( Lawsonia) Preparation of attenuated Salmonella mutant strain for simultaneous prevention or treatment of swine proliferative ileitis and Salmonellosis comprising amplifying any one gene selected from the group consisting of genes encoding OptA, OptB, FliC and Hly antigens derived from intracellularis ) Provide a method.

The present invention also provides an attenuated Salmonella mutant mixture comprising two or more of the mutants.

In addition, the present invention provides a vaccine composition for the simultaneous prevention or treatment of swine proliferative ileitis and Salmonellosis comprising the attenuated Salmonella mutant strain or a mixture thereof as an active ingredient.

In addition, the present invention provides a feed additive for the simultaneous prevention of pig proliferative ileitis and Salmonellosis comprising the attenuated Salmonella mutant strain or a mixture thereof as an active ingredient.

The attenuated Salmonella mutant strain of the present invention has an outer shell structure and shell structure similar to that of the Salmonella spp., And expresses the Lawsonia intracellularis-derived OptA, OptB, FliC or Hly antigen to the outside of the cell, thereby humoral and cellular Since it is possible to induce a mediated immune response, the mutant strain of the present invention is expected to be useful as a vaccine that can simultaneously prevent and treat swine proliferative ileitis and salmonellosis, which can be safely, economically and easily inoculated. In addition, the rfaL of the present invention Gene-deleted Salmonella mutants not only enhance the expression of selected Lawsonia antigens outside the cell, but also expand the availability of Salmonella probiotic vaccines, which have been limited in use due to misdiagnosis of serological tests using antibodies with LPS antigen. It is expected to do.

FIG. 1 is a simplified illustration of gene conversion when DNA fragments prepared with pKD3 as a template and pKD46 were transformed into a Salmonella strain ( S. Typhimurium ) having a target gene.

FIG. 2 is a result of comparing the surface O-antigen expression of JOL912, a strain containing O-antigen synthesis-related rfaL gene, and JOL1800, a strain lacking O-antigen synthesis-related rfaL gene, and is a result of silver staining.

Figure 3 is a result of confirming the degree of cell death of wild-type strains JOL401, mutant JOL912 and JOL1800 through the complement-dependent cytotoxicity test using rabbit serum complement.

Figure 4 shows the invasion / phagocytosis (4 hours, 24 hours, 48 hours after infection) of the Salmonella strains. Blue is macrophage, fluorescent green is Salmonella strain ( S. Typhimurium ). A) JOL401, B) JOL912, C) JOL1800.

5 is a result of confirming the serum IgG response to LPS of Salmonella strains (JOL401, JOL912, JOL1800). Displays the relative value measured at OD ₄₉₂ .

6 is a result of Western blot analysis of each Lawsonia protein expressed in JOL1800-derived strains (JOL1809, JOL1810, JOL1811 or JOL1812) expressing each Lawsonia antigen. Arrows indicate the expected size of each protein. M) Marker, A) Periplasmic sample of control strain, A ') Supernatant sample of control strain, B) Interspace sample of OptA expressing strain JOL1809, C) OptB Intercellular sample of the expression strain JOL1810, C ') Supernatant sample of the OptB expression strain JOL1810, D) Intercellular sample of the FliC expression strain JOL1811, E) Intercellular sample of the Hly expression strain JOL1812, E') Hly expression Supernatant samples of strain JOL1812.

Fig. 7 shows the result of confirming the titer of IgG for each antigen in the mouse group immunized by mixing four kinds of JOL1800-derived strains expressing each Lawsonia antigen. Comparison of non-immunized mice (Group A, white bars) and mice immunized with all four antigens (Group J, gray bars). I) IgG that specifically responds to OptA, II) IgG that specifically responds to OptB, III) IgG that specifically responds to FliC, IV) IgG that specifically responds to Hly. * Marked when there is the largest significant difference between control and immunized groups ( P <0.02).

Fig. 8 shows the results of confirming the titer of secreted IgA for each antigen in the mouse group immunized by mixing four strains of JOL1800 expressing each Lawsonia antigen. FIG. 8A shows the vaginal mucosal secretion IgA (viginal sIgA), and FIG. 8B shows the titer of intestinal secretion IgA (intestinal sIgA). Non-immunized mice (Group A, white bars), mice immunized with all four antigens (Group J, gray bars). V, IX) IgA, VI, X) Responsive to OptA, IgA, VII, XI) IgA, VIII, XII) Responsive to Hly IgA. * Marked when there is the largest significant difference between control and immunized groups ( P <0.02).

Figure 9 shows the results of the T cell immune response of splenocytes isolated from mice using FACS. I) Comparison of T cell changes of CD3 ⁺ CD4 ⁺ T cells and CD3 ⁺ CD8 ⁺ in non-immunized and immunized mice. II) The extent of CD3 ⁺ T cells, CD3 ⁺ CD4 ⁺ T cells and CD3 ⁺ CD8 ⁺ T cells increase in immunized mice against non-immunized mice. Non-immunized mice (A) and mice immunized with a mixture of four strains derived from JOL1800 expressing each Lawsonia antigen (JOL1800).

FIG. 10 shows mRNA expression results of cytokine genes measured by re-stimulation of splenocytes isolated from mice immunized by mixing immunized mice with four strains of JOL1800 expressing each of the Lawsonia antigens. The amount of cytokine expressed in non-immunized mouse group A was increased to 1 relative expression level. The spleen cells of mice belonging to group J were divided and stimulated with OptA, OptB, FliC, and Hly, respectively, and the cytokine expression levels were measured.

In order to achieve the object of the present invention, the present invention provides an attenuated Salmonella mutant comprising any one antigen selected from the group consisting of OptA, OptB, FliC and Hly antigens derived from Lawsonia intracellularis .

The Lawsonia Intracellularis (hereinafter L. intracellularis Gram-negative intracellular parasites are known as causative agents of swine proliferative ileitis.

Synthesis of LI0649 (802639-805194) and flagellin encoding autotransporter proteins from NCBI's Lawsonia intracellular PHE / MN1-00 gene map to select Lawsonian antigens of the present invention The genomic sequences of LI0570 (701958-702842) and hook HE0004 (3454-4209) genes encoding hook associated flagella C were identified. To determine the vaccine antigen suitability of each gene, the epitope of the antigenic protein was determined using the ProteomeBinders Epitope Choice Resource (http://bioware.ucd.ie/epic/) program. Antigen determinants for B cell expression for humoral immune responses directly bind to water-soluble antibodies, so the ratio of hydrophilicity residues is considered, and antigenicity factors are predicted by protein structure. The site with high function as a determinant was predicted.

Autotransporter (Opt), one of the Gram-negative bacteria outer membrane proteins, is an N-terminal signal peptide, a passenger domain, and a β-domain. The N-terminal signal peptide transports the protein synthesized in the passenger domain to the cytoplasmic membrane and expresses the protein transported by the β-domain to the cytoplasmic membrane to the outer membrane. It creates a path that can be made. These expressed passenger domain proteins are known to play a role in bacterial adhesion to the host's target organs or cells, invasion, and bacterial virulence, but have been attempted in live vaccines to prevent PPE. Never

The OptA and OptB antigens are 101-200 including a portion of the first site, the passenger domain, which is expected to be the most antigenic of the LI0649 genes (802639-805194) encoding the Lawsonia intracellularis derived autotransporter protein. The first amino acid sequence portion was selected as the first candidate protein (OptA), and the 534-851th amino acid sequence portion including the portion of the passenger domain and the entirety of the β-domain was determined as the second candidate protein (OptB).

The range of OptA antigens according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 1 and functional equivalents thereof. "Functional equivalent" means at least 60%, preferably at least 80%, more preferably at least 90%, even more preferably at least 60% of the amino acid sequence represented by SEQ ID NO: 1 as a result of the addition, substitution, or deletion of the amino acid Refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 1. "Substantially homogeneous physiological activity" means swine proliferative ileitis vaccine activity. The invention also includes fragments, derivatives and analogues of the OptA antigen.

The range of OptB antigens according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 2 and functional equivalents thereof. Details of the functional equivalents of the protein are as described above. Also included are fragments, derivatives and analogues of OptB.

The FliC antigen was selected from a gene encoding hook associated flagella C (Lasonia PHE / MN gene map LI0570 gene) that synthesizes flagellin, a protein constituting flagella. . Flagella on the cell surface that regulates bacterial movement usually acts as pathogen-associated molecular patterns (PAMPs) to activate the acquired immune system as a component of the innate immunity in the host. It interacts with Toll like receptor 5 (TLR5), one of the stimulating factors, and activates NF-κB in macrophage and contributes to the activation of the acquired immune system. However, the flagella gene has not yet been used as a candidate gene for PPE live vaccines.

Lawsonia intracellularis has one long flagella, and in this study, the protein associated with the flagella hook was selected as the antigen. The site of selection of the antigen is the flagellin N-terminal helical portion and the C-terminal spiral (C-terminal helical), which are conserved regions of 885nt (701958-702842). It may be more effective in inducing immune responses due to PAMPs, including the moiety.

The scope of FliC according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 3 and functional equivalents thereof. Details of the functional equivalents of the protein are as described above. Also included are fragments, derivatives and analogues of FliC.

The Hly antigen of the present invention was selected from Lawsonia intracellularis derived hemolysin (Hly) gene (LI0004 gene of the Lawsonia PHE / MN gene map). Lossonia intracellular lyse breaks down the entry vacuole that forms when it enters the cytoplasm and escapes from the host's phagolysosomal fusion. Hemolysin releases membrane-damaging cytolytic enzymes. It is known to produce and help break down inlet vesicles. On the 5th day of infection, the vesicles escaped from the vacuoles in the small intestine villus and gland cells (crypt cells) and grew freely in the cytoplasm.

The Hly antigen selection site synthesizes a hemolysin A protein known as bacterial pore forming toxin at a size of 756 nt (3454-4209). As a virulent factor, the hemolysin gene has been widely used as a candidate gene for vaccine development of other bacteria, but it has not been used as a candidate gene for PPE live vaccine.

The range of Hly according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 4 and functional equivalents thereof. Details of the functional equivalents of the protein are as described above. Also included are fragments, derivatives and analogues of Hly.

The attenuated Salmonella mutant strain of the present invention may be deleted asd gene. Preferably Salmonella mutant strains lon, cpxR and asd genes are deleted, more preferably lon, cpxR , rfaL And Salmonella mutants lacking the asd gene, but are not limited thereto.

In Salmonella mutants according to an embodiment of the present invention, Salmonella mutants ( Δlon) in which lon, cpxR and asd genes are deleted ΔcpxR Δasd Salmonella Typhimurium (JOL912) is a DAP (diaminopimellic acid) requester that lacks the asd gene, and is designed to select antigen-recombinant strains without antibiotics.In addition, by deleting cpxR , lymphocyte penetration is increased to increase immunogenicity, lon Deletion of the gene can attenuate pathogenicity. It is known that the strain has no effect on the production of extracellular polysaccharide (EPS), which can act as an antigen, and thus acts as an antigen by itself, resulting in sufficient humoral mucosal cellular immune response.

In one embodiment of the present invention, in order to increase the expression of foreign antigens in the existing S-type attenuated Salmonella strain (JOL912), the gene involved in the expression of Salmonella lipopolysaccharide (LPS) was progressed. . LPS is the part that forms outer membrane in Gram-negative bacteria, and removes rfaL gene involved in the synthesis of O-antigen polysaccharide (O-PS) polymerase of LPS to reduce the length of LPS synthesized in outer membrane. Made short. The O-antigen free rough Salmonella mutants ( Δlon) ΔcpxR Δasd ΔrfaL , JOL1800) maintains the antigenicity of LPS and exerts an immune response stimulated by external antigens by exposing the host's immune system to obscuring the LPS during the expression of the extracellular membrane of the selected Losonia intracellulase antigen gene. Could be augmented.

According to one embodiment of the invention, JOL1800 survived in the spleen of the host to effectively penetrate macrophages in mice and induce an immune response regardless of the inoculation route. However, serum complement was more easily killed than attenuated Salmonella strain derived from wild strain (JOL912). The attenuated JOL1800 eliminates O-antigens is more sensitive to serum complement but is thought to complement the weakened defense mechanism by avoiding LPS-specific antibodies in individuals that have already been exposed to Salmonella strains.

An additional benefit of using the Salmonella strain from which the O-antigen has been removed as a delivery vector is the Differentiation of Infected and Vaccinated Animals (DIVA). JOL1800, a strain produced by completely removing O-antigen, a repeating sugar polymer constituting the outermost part of LPS, was used as a vector to transfer heterologous antigens. It can be used in DIVA to distinguish between infected individuals and individuals immunized with vaccine strains lacking O-antigens.

The attenuated Salmonella strain of the present invention is any one of the genes selected from the group consisting of Bla (β-lactamse) signal sequence, genes encoding OptA, OptB, FliC and Hly antigens linked thereto; And asd gene; but may be transformed with a recombinant vector comprising, but is not limited thereto. In one embodiment of the invention, the recombinant vector may be pJHL65 (asd + vector, pBR ori, 6xHis) or pJHL80 (asd + vector, p15A ori, 6xHis) having a secretion system based on the Bla signal sequence.

Further, in the Salmonella mutants of the present invention, wherein the Salmonella is Salmonella typhimurium (Salmonella typhimurium), Salmonella tie blood (Salmonella typi), Salmonella para tie blood (Salmonella paratyphi), Salmonella Sendai (Salmonella sendai), Salmonella Galina Solarium ( Salmonella gallinarium ) or Salmonella enteritidis ( Salmonella enteritidis ) and the like, preferably Salmonella typhimurium, but is not limited thereto.

In addition, the present invention

(a) Lawsonia Intracellulose amplifying any one gene selected from the group consisting of genes encoding OptA, OptB, FliC and Hly antigens derived from intracellularis );

(b) cloning the amplified gene of step (a) into a recombinant vector having an asd gene;

(c) transforming the cloned plasmid of step (b) into an attenuated Salmonella strain; And

(d) provides a method for producing attenuated Salmonella mutant strain for simultaneously preventing or treating swine proliferative ileitis and Salmonellosis comprising the step of selecting the transformed Salmonella mutant strain of step (c).

Salmonella mutant mixture of the present invention is lon, cpxR , rfaL And asd Salmonella mutants having a gene deleted are mixtures containing two or more Salmonella mutants expressing any one antigen selected from the group consisting of OptA, OptB, FliC and Hly antigens in the extracellular membrane or extracellular.

Preferably, the mixture of Salmonella mutants may be a mixture comprising all of Salmonella mutants expressing OptA, Salmonella mutants expressing OptB, Salmonella mutants expressing FliC and Salmonella mutants expressing Hly, but are not limited thereto.

The vaccine composition of the present invention is a Lawsonia intracellular cellulose to Salmonella attenuated by gene deletion (lawsonia) Intracellularis ) containing a mixture containing one or more Salmonella mutants expressing OptA, OptB, FliC and Hly antigens as an active ingredient, by treating the vaccine composition in humans or animals to prevent or prevent pig hyperplasia ileitis and Salmonellosis It can be cured.

In the vaccine composition according to an embodiment of the present invention, the Salmonella mutant strain mixture may be prepared in the form of mutant strains or dead bacteria, preferably in the form of mutant strains, but is not limited thereto.

The composition of the present invention may be administered orally or parenterally (eg, applied intramuscularly, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, preferably orally or subcutaneously. This is not restrictive. In addition, the dosage of the composition varies depending on the weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of the disease or the like of the human or animal.

The vaccine composition may be inoculated in humans or mammals, and the mammal may be cows, deer, goats, goats, dogs, pigs, and the like, and preferably inoculated in pigs, but is not limited thereto.

As used herein, the term “vaccine” refers to a biological agent containing an antigen that immunizes a living body, and refers to an immunogen or antigenic substance that immunizes the living body by injection or oral administration to a human or animal for the prevention of infection. In vivo immunization is largely divided into automatic immunity obtained automatically by the in vivo immunity after infection of a pathogen and passive immunity obtained by an externally injected vaccine. While autoimmunity is characterized by a long period of production of immune-related antibodies and continuous immunity, passive immunization with vaccines acts immediately to treat infectious diseases, but has a disadvantage of poor sustainability.

The vaccine composition includes stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylene, subviral particle adjuvants, cholera toxin , N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium bromide and mixtures thereof The second adjuvant may be further contained.

In addition, the vaccine composition may comprise a veterinary acceptable carrier. As used herein, the term "veterinary acceptable carrier" includes any and all solvents, dispersion media, coatings, antigen adjuvant, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Carriers, excipients, and diluents that may be included in the composition for vaccines include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

In addition, the vaccine composition is an oral formulation such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and nasal formulations such as drips or sprays and sterile injectable solutions, respectively, according to a conventional method. Formulated in the form of can be used. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used can be prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate and sucrose in the lecithin-like emulsifier. Or lactose, gelatin, etc. can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc may also be used. As a liquid preparation for oral administration, suspending agents, liquid solutions, emulsions, syrups, etc. may be used.In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be used. May be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations. As the non-aqueous preparation and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like may be used, but are not limited thereto. Suitable penetrants for formulations for intranasal administration are generally known to those skilled in the art. Such suitable formulations are formulated to be preferably sterile, isotonic and buffered for stability and compliance. Formulations for intranasal administration are also formulated to stimulate mucus secretion in several aspects to maintain normal ciliary action, and suitable formulations are preferably slightly buffered formulations that maintain isotonicity, pH 5.5 to 6.5, and most preferably Antimicrobial preservatives and suitable drug stabilizers.

The feed additive of the invention rosoni ah intra-cell-less (Lawsonia intracellularis) derived OptA, OptB, including a Salmonella mutant, or a mixture thereof to express and secrete FliC and Hly antigen as an active ingredient, a Salmonella mutant, or a mixture thereof rosoni O And as well as the protective effect on Salmonella, effectively induces cellular and humoral immune response in animals, when used as feed additives can contribute to the simultaneous prevention and immune boosting of pig proliferative ileitis and Salmonellosis in livestock.

The feed additive of the present invention may use the Salmonella mutant mixture as it is or additionally add known carriers, stabilizers and the like, such as grains and by-products allowed for livestock, citric acid, fumaric acid, adipic acid, lactic acid, if necessary. , Organic acids such as malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechins, alpha-tocopherols, rosemary extracts, vitamin C, green tea extracts, licorice extracts, chitosan, Natural antioxidants such as tannic acid and phytic acid, antibiotics, antibacterial agents, and other additives may be added, and the shape may be in a suitable state such as powder, granules, pellets, suspensions, and the like. It may be supplied alone or mixed with the feed.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

재료 및 방법Materials and methods

1. O-항원 결손 약독화 살모넬라 균주 개발1. Development of attenuated Salmonella strain of O-antigen deletion

1.1. 실험에 사용된 균주 및 플라스미드1.1. Strains and Plasmids Used in the Experiment

The strains and plasmids used in the present invention are shown in Table 1. Salmonella typhimurium (hereinafter, referred to as S. Typhimurium ) strain from which the asd gene was removed was cultured by adding 50 µg / ml DAP at 37 ° C in LB medium. Temperature sensitive strains were cultured in LB medium, 28 ° C. Strains with lambda-red genes were cultured in LB medium containing 0.2% L-arabinose. All strains were stored at −80 ° C. in LB liquid medium containing 20% glycerol.

1.2. 실험동물1.2. Laboratory animals

All experimental animal care and procedures described in the present invention were approved by Chonbuk National University Animal Ethics Committee (CBNU2015-00085) in accordance with the guidelines of the Korean Committee on Animal Care. BALB / c female mice of 5 weeks of age were purchased and used in experiments after a period of adaptation for about 1 week while being bred at the experimental animal breeding center of Chonbuk National University.

1.3. O-항원 결실 변이주의 제작1.3. Production of O-antigen Deletion Mutantism

The O-antigen ligase gene, rfaL, was removed from the existing attenuated S. Typhimurium to develop a strain without O-antigen in LPS on the bacterial surface. Plasmid pKD46 containing lambda red, which induces recombination, was transformed by electric shock into S. Typhimurium JOL912 ( ΔcpxR, Δlon, Δasd ) , which is attenuated live bacteria. After culturing at 32 ° C. or lower (28-30 ° C.), PCR was confirmed whether the plasmid was successfully introduced. The pKD3 plasmid as a template was amplified by PCR using a 40-nucleotide-long primer containing the rfaL gene and a portion of the pKD3 plasmid. The amplified PCR product was extracted and transformed into S. Typhimurium competent cells into which pKD46 was introduced. It was plated on LB (Luria-Bertani) solid medium to which arabinos was added and incubated at 37 ° C. Recombined strains were selected in LB solid medium containing 25 μg / ml chloramphenicol. Knock-out was confirmed by PCR using primers. In order to remove the antibiotic resistance gene, competent cells were made from colonies confirmed to be transgenic, and the pCP20 plasmid was transformed. Cultured in LB medium, 37 ℃ was selected bacteria without chloramphenicol resistance. LPS was extracted and confirmed by SDS-PAGE for comparison with wild S. Typhimurium . The finally identified O-antigen deleted Salmonella strain was named JOL1800.

1.4. 인 비트로 보체 민감성 분석(In vitro complement sensitivity assay)1.4. In vitro complement sensitivity assay

Serum complement sensitivity of S. Typhimurium was measured using rabbit serum. Serum free of anti-salmonella antibodies was taken from rabbits and used for analysis. All strains used were incubated until late log phase and diluted to 1 × 10 ⁷ cfu / 100 μl. The prepared JOL401, JOL912, and JOL1800 cultures were respectively mixed with 100 μl PBS, 100 μl of 50% serum complement, and 100 μl of complement-free serum (DCS), respectively, and incubated at 37 ° C. for 1 hour. All cultures were then plated in different LB solid medium and cultured. DAP was added to the medium in which JOL912 and JOL1800 grew. The experimental results were expressed as% of inoculum = (CFU _treatment / CFU _PBS ) × 100.

1.5. 대식세포 탐식/침투 분석1.5. Macrophage Phagocytosis / Infiltration Analysis

The primary peritoneal macrophage of mice was infected with JOL401, JOL912, and JOL1800, and the phagocytosis and infiltration were observed using a fluorescence microscope. Macrophages were harvested by injecting 5% BSA into the abdominal cavity of 4 week old mice. Macrophages were treated aseptically and covered with 0.2% gelatin-coated coverglass slip in a 5 × 10 ⁶ cell number. Thereafter, RPMI [RPMI 1640, 10% FBS (heat-inactivated fetal bovine serum), 1 × Antibiotic-Antimycotic (Gibco, Life technologies, USA)] was poured into 6-well cell culture plates. After 24 hours of incubation in an incubator at 37 ° C., 5% CO ₂ , S. Typhimurium was infected with MOI 10: 1. After bacterial treatment, the cells were incubated in 37%, 5% CO ₂ incubator for 30 minutes. The phagocytic or non-penetrating bacteria were washed with PBS and placed in the incubator for 48 hours with the addition of RPMI containing antibiotics. Cover glass slip cells were fixed with 3% paraformaldehyde diluted with PBS and subjected to immunofluorescence staining. The primary antibody is chicken anti-912 polyclonal sera when infected with JOL401, JOL912, or chicken anti-1800 polyclonal sera when infected with JOL1800. Each diluted 1: 1,000 in 0.1% tween 20 PBS was used. All secondary antibodies were used diluted in PBS with goat anti-chicken IgY H & L-Alexa Fluor® 488 (Abcam, UK) at a ratio of 1: 5,000. The nuclei of macrophages were counterstained at a concentration of 0.5 μg / ml DAPI (2- (4-amidinophenyl) -1H-indole-6-carboxamidine; Sigma-Aldrich, US). Cover glass slip was attached to a micro-slide and observed with a fluorescence microscope (AX1 Zeiss., Germany). Macrophages were observed 4 hours after infection, 24 hours after and 48 hours after infection.

1.6. 살모넬라 균주의 생존 분석1.6. Survival Analysis of Salmonella Strains

S. Typhimurium strain of the present invention was carried out to determine how long the remaining in the host organs. A total of 96 rats were divided into three groups (n = 32). Four-week-old mice were orally inoculated with JOL401 and JOL1800 at 1 × 10 ⁷ cfu, respectively, and the third group was intramuscularly inoculated with JOL1800. For the purpose of the experiment, the Δasd strain JOL1800 was inoculated by transforming the asd + plasmid pJHL65 by electric shock. Spleens were harvested aseptically by anesthetizing 8 mice of each group every 3, 7, 14, 21, 28 days after inoculation. To confirm the presence of Salmonella in the spleen, the spleen was homogenized with 2 ml BPW (buffered peptone water, Becton, MD, USA) using a homogenizer. 100 μl of the homogenized solution was immediately inoculated evenly into Brilliant green agar (BGA) plates and incubated overnight at 37 ° C. Simultaneously, the remaining BPW samples were inoculated in Rappaport-Vassiliadis (RV) medium and incubated at 37 ° C. for 48 hours. The colonies obtained were finally S. Typhimurium PCR was confirmed using specific primers.

1.7. 혈청 IgG에 기초한 DIVA 능력 확인1.7. Identification of DIVA Ability Based on Serum IgG

A total of 60 mice are divided into 4 groups (n = 15). The first inoculation was carried out when the mice are 4 weeks old, and the second dose was given 3 weeks later. JOL401 and JOL1800 were used as the inoculating strains, and 1 × 10 ⁸ cfu / 100 μl was orally inoculated and 1 × 10 ⁶ cfu / 100 μl was intramuscularly inoculated. Serum was collected at weekly intervals up to 5 weeks after inoculation. Indirect ELISA was performed using purified S. Typhimurium LPS (L6511 SIGMA, sigma-Aldrich Co. LLC, US). Purified antigenic protein, LPS, was coated on an ELISA plate by dispensing at a concentration of 200 ng / well. Mouse serum, the primary antibody, was diluted 1: 100 with PBS, and HRP-condensed anti-mouse IgG, the secondary antibody, was diluted at a ratio of 1: 8,000. For the color development, 100 μl per well of the OPD (Sigma-Aldrich, US) reaction solution was dispensed for 5 minutes and then stopped with 3M H ₂ SO ₄ , and the OD value was measured at 492 nm. The value of serum IgG attached to LPS was expressed as the average OD value.

2. 2. L. intracellularisL. intracellularis 항원을 발현하는 백신 균주의 개발 Development of vaccine strains expressing antigen

2.1. 균주 및 플라스미드2.1. Strains and Plasmids

The bacterial strain, plasmid used in the present invention is Listed in Table 1. Commercially available vectors for expression of E. coli proteins, pET28a and pET32a plasmids have kanamycin resistance, so the strains were cultured in the presence of kanamycin (50 µg / ml). The Bl21 (DE3) pLysS strain was used for purifying Lawsonia antigen protein. The strain was cultured in the presence of 0.1M IPTG (Isopropyl β-D-1-thiogalactopyranoside) when inducing overexpression during protein purification. pJHL65 and pJHL80 asd + plasmids were used as vectors to secrete and deliver antigen proteins cloned in a Bla signal sequence-based periplasmic secretion manner. All strains were incubated at 37 ° C. using LB medium and stored at −80 ° C. using LB liquid medium containing 20% glycerol. DAP is at a concentration of asd 50㎍ / ㎖ - was added to culture the strain JOL1800.

2.2. 항원 단백질을 발현하는 플라스미드 제작2.2. Construction of Plasmids Expressing Antigen Proteins

Candidate antigens of L. intracellularis are OptA and OptB, which are involved in autotransporter synthesis, FliC, which synthesizes flagella related proteins, and Hly, which is considered to be involved in cell infiltration. Genes required for the expression of antigenic proteins were all obtained using gene synthesis (Bioneer, Korea). The genes of L. intracellularis that synthesize each protein were examined through GenBank of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). Each L. intracellularis antigen was analyzed by Peptide Property Calculator software (http://www.biosyn.com/peptidepropertycalculator/), and then synthesized by selecting the nucleotide sequence of the high antigenicity. The genes were cloned into the pBHA vector, with the restriction sites identified at each end. The restriction sites added at both ends of the L. intracellularis antigen gene are OptA for Sal I and Xho I, OptB for Sal I and Pst I, FliC for EcoR I and Sal I, and Hly for EcoR I and Hind III. Recombinant plasmids pET32a-OptA, pET28a-OptB, pET28a-FliC, and pET28a-Hly were prepared by cloning into the plasmids pET28a and pET32a, respectively, using the designated restriction enzymes. OptA was not inserted into pET28a and cloned into pET32a, a similar strain of plasmid. The resulting recombinant plasmid was transformed into E. coli BL21 (DE3) pLysS by a thermal shock method to prepare a strain for protein purification overexpressing the antigenic protein in the presence of IPTG. L. intracellularis inside strains The presence or absence of the antigen gene was confirmed by electrophoresis and PCR performed on agarose gel after plasmid was isolated from BL21 (DE3) pLysS, digested with restriction enzymes corresponding to each antigen. Primers used for Lawsonia insertion antigen identification PCR are listed in Table 2. The final identified strains were designated as JOL1593 (BL21 containing pET32a and expressing OptA), JOL1586 (BL21 containing pET28a and expressing OptB), JOL1682 (BL21 containing pET28a and expressing FliC), JOL1742 (BL21 containing pET28a and expressing Hly). OptA, OptB, FliC, and Hly proteins in each strain were purified using Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Valencia, CA). The antigenic protein of L. intracellularis obtained by culturing and purifying each strain was used to detect specific antibodies against each antigen in mice.

To prepare candidate vaccines for the simultaneous prevention of L. intracellularis and S. Typhimurium , the antigen genes were cloned into pJHL65 and pJHL80 plasmids that express heterologous antigens in the intercellular space, and then pJHL80-OptA, pJHL65-OptB, pJHL65-FliC. pJHL65-Hly was obtained.

2.3. 백신 균주 제작2.3. Vaccine strain production

PJHL80-OptA, pJHL65-OptB, pJHL65-FliC, and pJHL65-Hly prepared in 2.2 were transformed into JOL1800 by electric shock, respectively. The JOL1800 was washed twice with distilled water containing 10% glycerol, placed in a cuvette and mixed with 0.1 µg of purified plasmid, subjected to electric shock with Bio-Rad MicroPulser (Bio-Rad, USA), and recovered. LB liquid medium without DAP was recovered. 1 ml was incubated at 37 ° C. for 1 hour. To screen for transformed Salmonella bacteria, 100 μl of the culture solution was plated on LB agar without DAP and dried, and colonies formed after overnight culture were selected. Plasmids were recovered from the selected strains, digested with restriction enzymes corresponding to the respective antigens, and confirmed by electrophoresis and PCR on agarose gels.

2.4. 2.4. 웨스턴Weston 블롯Blot

The antigens of L. intracellularis expressed in the outer membrane and periplasmic space of the prepared vaccine strain were confirmed by Western blot. OptA in JOL1809, OptB in JOL1810, FliC in JOL1811 and Hly in JOL1812. Antigen proteins expressed in the recombinant strains were obtained in cell-free supernatants through TCA precipitation. To prepare the antigenic protein secreted by the recombinant plasmid, the cells were cultured at 37 ° C and LB liquid medium until the optical density (OD ₆₀₀ ) was measured at ₆₀₀ nm, and centrifuged at 3,400 × g for 20 minutes. The supernatant was then separated. 20% (v / v) trichloroacetic acid (TCA) solution was added and reacted overnight at 4 ° C. to find the protein secreted from the clear supernatant obtained by passing the supernatant through a 0.22 μm filter. The reacted supernatant was centrifuged at 15,700 × g for 30 minutes, and then the settled pellet was washed with acetone and resuspended in PBS. The resulting solution was used in a 1: 1 mixture with SDS-PAGE sample buffer. For comparison, an attenuated Salmonella strain with pJHL65 vector was used as a control in the same manner. Each protein sample was separated by SDS-PAGE for Western blot analysis and transferred to a 0.2 μm PVDF membrane (Millipore, Billerica, Mass., USA) at 4 ° C. with blocking buffer (3% BSA, PBS, 0.1% Tween-20). The reaction was overnight. The next day, the anti-his-tag antibody (IG Therapy Co., Ltd., Korea) was diluted to 1: 5,000 and reacted for 1 hour, followed by HRP (horseradish peroxidase) -condensed goat anti-mouse IgG diluted to 1: 5,000. Time was processed. The results were stained using a WEST-ZOL Plus Western Blot Detection System (iNtRon, Korea) and the expression of each antigen was confirmed using a multi-wave length illumination system KODAK Image Station 4000MM (Kodak, USA).

3. 3. L. intracellularis L. intracellularis 항원 종합 실험Antigen synthesis experiment

3.1. 균주 및 플라스미드3.1. Strains and Plasmids

The strains and plasmids used in this experiment were recombinant attenuated Salmonella strains JOL1809, JOL1810, JOL1811, and JOL1812, which express the L. intracellularis antigens in Table 1.

3.2. 실험동물3.2. Laboratory animals

In order to confirm the immunogenicity of L. intracellularis OptA, OptB, FliC, and Hly antigens, 16 mice were divided into two groups and used in the experiment (n = 8). The following conditions are the same as 1.2.

3.3. 백신 준비3.3. Vaccine Preparation

The O-antigen-deficient attenuated Salmonella strains expressing L. intracellularis antigens OptA, OptB, FliC, and Hly, respectively, were grown in LB solid medium and JOL1809, JOL1810, JOL1811, and JOL1812 were inoculated in LB liquid medium. Incubated overnight at ° C. Separately cultured strains were added to the LB liquid medium to be diluted at a ratio of 1: 100, respectively, and incubated for 3-5 hours until the OD ₆₀₀ value was 0.6. The culture was centrifuged at 190 × g at room temperature and the precipitate was washed three times with sterile PBS. After the final precipitate was suspended in sterile PBS again, the bacterial count was measured by checking the OD ₆₀₀ value. JOL1809, JOL1810, JOL1811, and JOL1812 strains were mixed by 5 × 10 ⁷ CFU, respectively, and a total of 2 × 10 ⁸ CFU / 100 μl strains were determined as the final inoculation amount.

3.4. 백신 접종3.4. vaccination

Vaccination was performed to confirm the induction of immune response when four inoculations of OptA, OptB, FliC, and Hly of L. intracellularis were inoculated. Mice were divided into two groups (n = 8), and one group was subcutaneously inoculated with sterile PBS, and the other group was mixed with 5 × 10 ⁷ CFU of JOL1809, JOL1810, JOL1811, and JOL1812, respectively. 10 ⁸ CFU / 100 μl strains were subcutaneously inoculated.

3.5. 가검물 채취3.5. Specimen collection

Blood was collected for vaginal lavage, intestinal lavage, and IgG for sIgA measurements before and after inoculation. In the case of vaginal washing solution, the solution was stored at -20 ° C after vaginal washing using PBS and used for the experiment. Intestinal lavage was performed based on the pilocarpine-based lavage method. Prepare wash solution (Lavage base 4ml, polyethylene glycol 6.5g, D.W.40ml) and feed 500µl per animal, and inject 20% of pilocarpine 0.5% per animal intraperitoneally. Each rat feces collected on the plate was treated with 500 μl of 50mM EDTA, centrifuged at 3,400 × g for 15 minutes, and the supernatant was separated and stored at -20 ° C. and used for the experiment. Serum was collected by orbital venous blood collection and centrifuged at 4,000 × g for 5 minutes to separate serum, which was supernatant, and stored at -20 ° C.

3.6. ELISA 분석3.6. ELISA analysis

ELISA was performed to measure sIgA and IgG specific for L. intracellularis OptA, OptB, FliC, and Hly antigens expressed in recombinant vaccine strains. As the coating antigen, OptA, OptB, FliC, and Hly antigen proteins purified from JOL1586, JOL1593, JOL1682, and JOL1742 strains were used. Purified antigenic protein was administered at a concentration of 500 ng / well, and standard protein wells were coated with goat-anti mouse IgG or goat anti-mouse sIgA at a concentration of 200 ng / well, respectively, and then coated overnight at 4 ° C. The coated plate was washed three times with PBS (PBST) containing 0.05% of Tween 20 and then blocked with blocking buffer (3% skim milk in PBS) at 37 ° C. for 1 hour. Serum and PBS were diluted 1: 100, vaginal lavage was diluted 1: 3, intestinal lavage was diluted 1: 4, and 100 μl was dispensed into the wells, followed by reaction at 37 ° C. for 1 hour. In the case of serum, goat anti-mouse IgG HRP and vaginal lavage were diluted 1: 5,000 in goat anti-mouse IgA HRP, and 100 μl was dispensed into each well, followed by reaction at 37 ° C. for 1 hour. 100 μl of the OPD-substrate reaction solution was dispensed per well, and then stopped in 3M H ₂ SO ₄ after color development. The OD value was measured at 492 nm. The concentration of each antigen specific antibody was determined based on the standard protein concentration.

3.7. T 세포 면역반응 측정3.7. T cell immune response

A group of control and immunized mice were prepared (n = 5), and 10 days after vaccination, the mice were sacrificed, the spleen was collected aseptically, crushed, the cells were taken out of the tissues, and the remaining tissues were removed with a cell strainer. Removed. The pellet was suspended by RPMI 1640 by centrifugation at 470 × g for 3 minutes, and then the cells were suspended using RBC elution buffer to remove RBC. Then washed twice with RPMI 1640 and the last pellet was suspended with RPMI. The number of cells suspended in RPMI was measured and aliquoted into 96 well plates to be 1 × 10 ⁶ cells / well. Fluorescence was performed for FACS (fluorescence activated cell sorting) analysis. Anti-mouse CD3e-PE, anti-mouse CD4-perCP-vio700, and anti-mouse CD8a-FITC were reacted at 4 ° C. for 15 minutes in dark conditions. Stained cells were washed three times with 200 μl FACS buffer and analyzed using the MACSQuant ^® analyzer (miltenyi Biotec, Germany).

3.8. 역전사 실시간 PCR을 통한 사이토카인의 mRNA 측정3.8. Cytokine mRNA Measurement by Reverse Transcription Real-Time PCR

Cells were divided into 96 well plates in the same manner as in 3.7. OptA, OptB, FliC, and Hly were stimulated at 200 ng / μl for 1 hour at 5% CO ₂ environmental conditions in 1x10 ⁶ cells for 48 hours. After incubation, total RNA was extracted using GeneAll ^® Hybrid-RTM kit and synthesized into cDNA using ReverTra Ace ^® qPCR RT Kit. Primers of mouse interferon-gamma (IFN-γ), interleukin (IL) -4, and IL-17 used for real-time PCR are listed in Table 3. Real-time PCR was measured by using ^{SYBR ® Green Real-Time PCR Master} Mix (QPK-201, TOYOBO, Japan). It was measured using a Step One plus Real Time PCR system (Applied Biosystems). For each cytokine, the amount of RT-PCR product was normalized to β-actin values using internal standards.

Primer used in RT-PCR of the present invention

항원antigen	프라이머 방향Primer direction	서열(5'→3')Sequence (5 '→ 3')	서열번호SEQ ID NO:
IFN-γIFN-γ	정방향Forward direction	TCA AGT GGC ATA GAT GTG GAA GAATCA AGT GGC ATA GAT GTG GAA GAA	1313
IFN-γIFN-γ	역방향Reverse	TGG CTC TGC AGG ATT TTC ATGTGG CTC TGC AGG ATT TTC ATG	1414
IL-4IL-4	정방향Forward direction	ACA GGA GAA GGG ACG CCA TACA GGA GAA GGG ACG CCA T	1515
IL-4IL-4	역방향Reverse	GAA GCC CTA CAG ACG AGC TCAGAA GCC CTA CAG ACG AGC TCA	1616
IL-17IL-17	정방향Forward direction	ACC GCA ATG AAG ACC CTG ATACC GCA ATG AAG ACC CTG AT	1717
IL-17IL-17	역방향Reverse	TCC CTC CGC ATT GAC ACATCC CTC CGC ATT GAC ACA	1818

3.9. 통계분석3.9. Statistical analysis

Statistical analysis was performed using STATA (Stata Statistical Software, Release 8.0, Stata Corporation, College Station, TX). Antibody titer data of the ELISA, CD3 +, CD4 +, CD8 + T cells, cytokine fold change (2 ^- ^ΔΔCT ) were considered significant for P <0.05. Unless otherwise specified, expressed as standard deviation (SD).

실시예 1. O-항원 결손 약독화 Example 1. Attenuation of O-antigen Depletion S.S. Typhimurium 균주 개발 Typhimurium strain development

1.1. 살모넬라 생백신 벡터의 제작1.1. Production of Salmonella Live Vaccine Vector

DIVA availableS. Typhimurium Live vaccine vector JOL1800 attenuates with high immunogenicity and permeabilityS. Typhimurium It was produced by improving the strain JOL912. JOL912 has three genes (Δlon ΔcpxR ΔasdAuxotrophic mutant strainsasd Deletion of the gene necessitates DAP upon replication. Thus, asd + plasmids are used as vectors expressing antigen genes to satisfy balanced-lethal complementation. Using Ramma Red Recombination, an additional O-antigen ligase generfaLIs deleted from JOL912, a new Salmonella vaccine vectorΔlon ΔcpxR Δasd ΔrfaL JOL1800 was produced (FIG. 1). The lambda RED plasmid pKD46 was transformed into JOL912 by electric shock, and the cat gene cassette amplified by PCR was introduced into the strain transformed with pKD46. The bacteriophage recombination system, called gamma-red, contains gamma, beta, and exo genes. Exo and Bet genes work together to promote gene recombination. Exo acts at both ends of the ds DNA to create a 3-overhang, and bet binds to 3 ss DNA, causing strand exchange by RecA. The cat gene cassette exchanged here made the plasmid pKD3 a template. When incubated at 37 ° C., JOL912 with pKD46 and linear DNA was introduced to replace rfaL with the cat gene cassette. At the same time pKD46 exits the cell. Removal of the target gene was confirmed by PCR using primers from the rfaL flanking region. When wild rfaL is changed to cat gene cassette (~ 1.1kb), the size of the amplicon, which is 1.9kb in total, is shortened to 1.7kb. Plasmid pCP20 was transformed into a strain in which removal of the rfaL gene was confirmed. pCP20 was introduced to remove antibiotic resistance genes and expressed FLP recombinase that acts directly on the FLP recognition target (FRT) sites present at both ends of the antibiotic resistance gene cassette. After culturing at 30 ℃ to obtain a strain from which the cat gene cassette was removed, and further cultured at 37 ℃ or 40 ℃ to obtain a final strain without the plasmid pCP20. The last strain obtained was not resistant to antibiotics derived from JOL912.lon, cpxR , asd , rfaL The mutant strain JOL1800 was deleted. JOL1800rfaL It is a rough Salmonella strain without O-antigen due to the gene removal. After PAGE separation and purified LPS extraction in JOL1800 it can be confirmed that there is no O-antigen on the surface of the bacteria (Fig. 2). In the S-type JOL912 used as a control, long-expressed O-antigens can be seen.

1.2. 인 비트로 보체 민감성 분석1.2. In Vivo Complement Sensitivity Analysis

To investigate the effect of O-antigen length on complement-dependent cellular injury, complement sensitivity analysis was performed by culturing wild S. Typhimurium JOL401, attenuated strain JOL912, and JOL1800, a strain derived from JOL912. R-type JOL1800 showed a more sensitive response to rabbit complement than the other S-type strains. JOL1800 showed a marked bacterial count reduction compared to wild JOL401 and JOL912 (FIG. 3).

1.3. 대식세포 탐식/침투 분석1.3. Macrophage Phagocytosis / Infiltration Analysis

Macrophages of mice were infected with O-antigen-deleted S. Typhimurium JOL1800 from wild S. Typhimurium JOL401, attenuated S. Typhimurium JOL912, JOL912 to identify differences in the ability of the three strains to feed or penetrate macrophages. .

As a result, JOL401, JOL912, JOL1800 all confirmed the infiltration of bacteria into macrophages 4 hours after infection. Salmonella have an infection pathway that survives in the macrophages of the host and spreads the bacteria systemically. Not only JOL912 attenuated by removing the lon, cpxR and asd genes, but also JOL1800, which additionally removed the O-antigen of the bacterial surface LPS by deleting the rfaL gene, was found to have the ability to invade and survive macrophages (FIG. 4). ).

1.4. 살모넬라 균주 생존 분석1.4. Salmonella Strain Survival Analysis

Mutant strains produced by the deletion of several genes are very attenuated and sometimes do not reach the real organs or die easily, sometimes causing infection. When attenuated strains are used as vaccines, proper attenuation control is required so that the vaccine strains do not survive for too long in the host to cause disease or to survive too short to cause an immune response. The mice were infected with JOL401, JOL912, and JOL1800 three days later, seven days later, 14 days later, and 21 days later. The spleens were collected and tested for the presence of bacteria (Table 4). All strains were detected in the spleen 3, 7, 14 days after inoculation. JOL401, JOL912 survived in the spleen until 21 days after inoculation. The recovery time of JOL1800 was 12 days and was investigated by PROBIT analysis. It can be seen that JOL1800 can invade the host's immune system and sufficiently induce an immune response.

1.5. 혈청 IgG에 기초한 DIVA1.5. DIVA based on serum IgG

ELISA was performed on the sera of mice inoculated with LPS extracted from S. Typhimurium to investigate the possibility of DIVA of the developed JOL1800 strain. The results after inoculation with JOL401, JOL912, and JOL1800 measured at OD ₄₉₂ were compared. If there is a significant difference in the measured result, it can be used for DIVA. S-type strains with O-antigens, JOL401 and JOL912, showed LPS-specific antibodies, whereas L-type strains without O-antigen were detected in JOL1800 (Fig. 5). Boosting was performed three weeks after the first inoculation, considering that LPS-specific antibodies could not be induced due to rapid in vivo clearance due to attenuation of JOL1800. However, there was no LPS specific antibody response after that and DIVA could be possible if JOL1800 was used as a vaccine. After 4 weeks of inoculation, ELISA was performed on the serum of each individual to expose only to naturally infected individuals exposed to S. Typhimurium with O-antigen and to vaccine strains lacking O-antigen due to the difference in absorbance values measured at OD ₄₉₂ . Identified objects.

실시예 2. Example 2. L. intracellularisL. intracellularis 항원을 발현하는 백신 균주의 개발 Development of vaccine strains expressing antigen

2.1. 백신 제작 및 개발2.1. Vaccine Production and Development

The plasmids pJHL80-OptA, pJHL65-OptB, pJHL65-FliC, pJHL65-Hly, which were prepared by cloning the synthesized L. intracellularis antigens OptA, OptB, FliC, and Hly into the antigen expression plasmids pJHL65 and pJHL80, do not have O-antigens. The vaccine strain was completed by electroshock to DIOL-capable attenuated S. Typhimurium strain JOL1800. pJHL65 and pJHL80 express the cloned antigen in the intercellular space or surface of the bacteria. Subsequently, the transformed plasmid was re-extracted, cut with the corresponding restriction enzymes, subjected to electrophoresis and PCR using agarose gel, and finally identified strains having the antigen gene were JOL1809 (OptA expression JOL1800) and JOL1810 (OptB expression JOL1800, respectively). ), JOL1811 (FliC expressing JOL1800), JOL1812 (Hly expressing JOL1800).

2.2. 개발된 균주에서 항원의 발현 유무2.2. Expression of antigen in the developed strain

Western blots were performed to confirm the expression of each antigen in JOL1809, JOL1810, JOL1811, and JOL1812. JOL1800 was used as a control for comparing antigen expression sites. The expression size of L. intracellularis antigen OptA was 17KDa, the expression size of OptB was 41.5kDa, the expression size of FliC was 38.6kDa, and the expression size of Hly was 30kDa (Fig. 6).

실시예 3. Example 3. L. intracellularis L. intracellularis 항원종합실험Antigen synthesis experiment

3.1. 전신과 점막에서의 면역 반응3.1. Immune response in the whole body and mucous membranes

L. intracellularis Antibody titers of IgG and sIgA were measured by ELISA in rats to assess the difference in humoral immune responses against the respective antigens when inoculated with JOL1800-derived S. Typhimurium strains expressing antigens OptA, OptB, FliC, and Hly. Serum IgG, vaginal mucosal sIgA, and mucosal IgA all showed antibody titer changes starting to rise and then up to about 4 weeks, reaching peak concentrations, and then decreasing (Figs. 7 and 8).

3.2. T 세포 면역반응3.2. T cell immune response

T lymphocyte subpopulation using FACS in splenocytes isolated from all immunized and non-immunized control mice to assess cellular immune responses following inoculation with recombinant attenuated S. Typhimurium live vaccines JOL1809, JOL1810, JOL1811, JOL1812 Was analyzed. Total T cells are expressed as CD3 ⁺ , and CD4 ⁺ and CD8 ⁺ are helper T cells and cytotoxic T cells, respectively. 9 days after the inoculation also CD3 ^⁺ CD4 ⁺ and CD3 ^⁺ CD8 ⁺ with the overall increase in the CD3 ⁺ It rose significantly (FIG. 9). CD3 ⁺ T cells showed an increase of 50% or more, CD4 ⁺ increased by 45% or more, and CD8 ⁺ increased by 55% or more compared to the control group.

3.3. 역전사 실시간 PCR을 통한 사이토카인의 mRNA 측정3.3. Cytokine mRNA Measurement by Reverse Transcription Real-Time PCR

RT-PCR was performed to measure the mRNA expression level of IFN-γ, IL-4, and IL-17 to evaluate the expression level of cytokines that regulate cellular immune responses. As a result of measuring cytokines by stimulating splenocytes of mice subcutaneously inoculated by mixing each strain expressing one Lawsonia antigen with each antigen, it was confirmed that IL-4 was significantly increased in OptA (FIG. 10). . Except for OptB, IL-4 and IFN-γ were significantly increased in all antigens. IL-17 is a cytokine secreted by differentiated Th17 cells, indicating that all antigens stimulate secretion. It is believed that CD4 ⁺ T cells have differentiated into Th1 and Th2 cells, and it is estimated that the differentiation of Th17 cells occurs.

Claims

Lawsonia Intracellular Solaris Attenuated Salmonella mutant strain comprising any one antigen selected from the group consisting of intracellularis ) OptA, OptB, FliC and Hly antigens.
The attenuated Salmonella strain of claim 1, wherein the OptA, OptB, FliC, and Hly antigens consist of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.
The method of claim 1, wherein the attenuated Salmonella mutant strain lon, cpxR , rfaL And an attenuated Salmonella mutant strain, characterized by the deletion of the asd gene.
According to claim 1, wherein said attenuated Salmonella strain is any one selected from the group consisting of genes encoding Bla (β-lactamse) signal sequence, OptA, OptB, FliC and Hly antigen linked thereto; And asd gene; attenuated Salmonella mutant strain, characterized in that transformed with a recombinant vector comprising a.
(a) Lawsonia Intracellulose amplifying any one gene selected from the group consisting of genes encoding OptA, OptB, FliC and Hly antigens derived from intracellularis );

(b) cloning the amplified gene of step (a) into a recombinant vector having an asd gene;

(c) transforming the cloned plasmid of step (b) into an attenuated Salmonella strain; And

(d) a method for producing attenuated Salmonella mutant strain for simultaneously preventing or treating swine proliferative ileitis and Salmonellosis comprising the step of selecting the transformed Salmonella mutant strain of step (c).
An attenuated Salmonella mutant mixture comprising at least two of the attenuated Salmonella mutants of claim 1.
Claim 1 attenuated Salmonella mutant strain or attenuated Salmonella mutant strain of claim 6 comprising a vaccine composition for the simultaneous prevention or treatment of pig proliferative ileitis and Salmonellosis.
According to claim 7, wherein the vaccine composition is a vaccine composition for the simultaneous prevention or treatment of pig proliferative ileitis and Salmonellosis, characterized in that oral or subcutaneous administration.
Claim 1 attenuated Salmonella mutant strain or attenuated Salmonella mutant strain of claim 6 comprising a feed ingredient for the simultaneous prevention of pig proliferative ileitis and Salmonellosis.