KR101893827B1 - Vaccine composition for preventing or treating porcine proliferative enteritis and salmonellosis simultaneouly comprising attenuated Salmonella mutant as effective component - Google Patents

Abstract

The present invention relates to a vaccine composition for the simultaneous prevention or treatment of porcine proliferative ileitis and salmonellosis comprising an attenuated salmonella mutant as an active ingredient. The invention rosoni ah intra-cell-less (Lawsonia intracellularis- derived OptA, OptB, FliC, or Hly antigens in a mouse inoculated with the transformed attenuated Salmonella mutant mixture, And cell - mediated immune responses were induced. Therefore, the mutant of the present invention is expected to be useful as a vaccine for the simultaneous prevention or treatment of swine-producing ileitis and salmonellosis which can be safely, economically and easily inoculated.

Description

Technical Field [0001] The present invention relates to a vaccine composition for preventing or treating simultaneous proliferative enteritis and salmonellosis, and a vaccine composition for preventing or treating simultaneous proliferation of salmonellosis and porcine proliferative ileitis comprising an attenuated salmonella mutant as an active ingredient.

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

Porcine proliferative enteritis (PPE) is a disease caused by Lawsonia intracellularis , which is prevalent all over the world and causes serious damage to swine farms. The worldwide incidence is estimated to be around 30%. In 2000, a survey of the National Animal Health Monitoring System (NAHMS) in the United States reported more than one-third of all farm households and 75% of large farm households. In Korea, it has been reported that more than 53% of infected individuals have been infected with the sample of pig farms nationwide.

PPE-induced diseases caused by thickening of the walls due to proliferation of intestinal cells (enterocyte) can be classified into proliferative hemorrhagic enteropathy and porcine intestinal adenomatosis type, and acute type porcine hemorrhagic intestine The disease occurs in pigs and shipped pigs recently stocked, and there are red and black tar lobe changes around the anus before the onset of the disease, and symptoms such as weakness, anemia and lack of appetite. The chronic form slows down feed intake, reduces body weight, and the main clinical symptoms are brown-colored, swollen diarrhea that lasts from a few days to four weeks. According to statistics PPE epidemic has been reported to cause massive economic losses to farmers, with a daily gain of 9 to 35 percent and a feed efficiency of 6 to 20 percent lower.

Absolute intracellular bacteria, which is an intracellular bacterium, is very difficult to cultivate in vitro and has a large variation in its growth rate compared to the epidemic rate and its seriousness in farms. Due to the limitations of the diagnosis and analysis of this disease, the development of detailed pathology as well as the development of treatment and diagnostic methods are limited. Conventional PCR methods and serologic diagnosis methods are used to diagnose the clinical symptoms of roundheads, but they are less susceptible to diagnosis of suspicious cases or host suspected cases. Because of this, it is difficult for the livestock farmers to know whether the disease is occurring on their own farm due to the nature of illnesses other than diarrhea. Antibiotics susceptible to intracellular growth-promoting Gram-negative bacteria such as tyrosine, tetracyclines, and licomycin have been used to prevent the disease. However, in Korea, The ban on antibiotics in feeds is increasing the need to develop an effective vaccine to control the germs present in pig farms.

In pigs with diarrhea, it is difficult to pinpoint the cause of the disease only by clinical symptoms or the form of diarrhea. In particular, diarrhea caused by fibrous necrotizing ulcerative colitis, which is caused by a typical swine diarrhea, pork salmonellosis with PPE, is mainly caused by Salmonella typhimurium . Salmonella, a Gram-negative bacterium that has proliferating properties in cells such as Lawsonia , has been shown to have a significant correlation with salmonella-bearing pigs, as well as with clinical symptoms, in recent studies. The study found that pigs infected with Rosacea bacillus increased the colonization of salmonella and promoted shedding due to the action of intestinal microbial guns in pigs. These two bacterial interactions suggest the urgency of developing a vaccine to prevent both diseases at the same time.

The number of B cells involved in humoral immune, the number of T cells involved in cellular immunity, and a significant decrease in SLAM7 (Signaling lymphocytic activation molecule), an expression receptor for T cells, were observed in pigs severely infected with Laosonia intracellularis. Reduced total lymphocyte counts and limited activation of cytotoxic T cells in these infected areas are known to be associated with proper environmental composition and subsequent modulation of immune responses to survive by L. intracellularis. In order to overcome the immunosuppressive reaction that may occur when PPE occurs, it is required to develop a vaccine having high antigenicity and stability.

On the other hand, Korean Patent No. 0758614 discloses the cultivation of Laussonia intracellularis, anti-rusonia intracellularis vaccine and diagnostic reagent, and in Korean Patent No. 1151004, A vaccine composition for the prevention and treatment of transformed attenuated Salmonella mutant and bovine coliform and Salmonella bacterium containing the same is disclosed. However, the present invention provides a vaccine composition for preventing and treating porcine proliferative ileitis comprising the attenuated salmonella mutant strain of the present invention as an active ingredient, There is no description about a vaccine composition for simultaneous prevention or treatment of salmonellosis.

The present invention has been made in view of the above-mentioned needs. The present inventors have found that, in order to simultaneously prevent the increase of the immune response due to the vaccine and the prevention of two types of porcine diarrhea, four kinds of antigens derived from rosa intracellulare (OptA, OptB, FliC, Hly) was cloned into a recombinant vector for a live bacterial vaccine containing the Bla signal sequence, transformed into LPS O-antigen deficient Salmonella strains, and humoral and cell-mediated expression of antigens in mice inoculated with the transformed Salmonella mixture Mediated immune response is induced, thereby completing the present invention.

In order to solve the above problems, 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 present invention also relates to a method of amplifying a gene selected from the group consisting of genes coding for OptA, OptB, FliC and Hly antigens derived from Lawsonia intracellularis , The present invention provides a method for producing attenuated Salmonella mutants for simultaneous prevention or treatment.

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 preventing or treating swine proliferative ileitis and salmonellosis which comprises the attenuated salmonella mutant or a mixture thereof as an active ingredient.

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

The attenuated Salmonella mutant of the present invention expresses OptA, OptB, FliC or Hly antigens derived from Rosacea intracellulase in an extracellular environment, having an outer component and an outer structure similar to those of the Salmonella outdoor strain, Mediated immune response, the mutant of the present invention is expected to be useful as a vaccine capable of simultaneously preventing and treating swine proliferative ileitis and salmonellosis which can be safely and economically and easily inoculated. In addition, the Salmonella mutant lacking the rfaL gene of the present invention can be used as an antimicrobial agent for improving the expression of the selected rossa antigen to the extracellular environment, It is expected to increase the availability of vaccines.

Fig. 1 is a brief diagram illustrating the transformation of a DNA fragment prepared using pKD3 as a template and a gene encoding pKD46 into S. typhimurium having a desired gene.
FIG. 2 shows the results of comparing the surface O-antigen expression of the smooth strain JOL912 and the rough strain JOL1800. The O-antigen of JOL912 and the surface O-antigen deficiency of JOL1800 were confirmed by silver staining.
FIG. 3 shows the result of confirming the degree of apoptosis of the wild-type strain JOL401, the S-type mutant JOL912 and the R-type mutant JOL1800 through a complement dependent cytotoxicity test using a rabbit serum complement. DCS; De-complemented serum.
Fig. 4 shows the appearance of Salmonella strains introgressing / phagocytosing in macrophages (4 hours, 24 hours, 48 hours after infection). Blue color is macrophage, fluorescent green is Salmonella strain ( S. Typhimurium). A) JOL401, B) JOL912, C) JOL1800.
Fig. 5 shows the result of confirming the serum IgG response to LPS of Salmonella strains (JOL401, JOL912, JOL1800). Relative value measured at OD ₄₉₂ is displayed.
FIG. 6 is a Western blot analysis result of each Rosonia protein expressed in a JOL1800-derived strain (JOL1809, JOL1810, JOL1811 or JOL1812) expressing each Rosaceae antigen. Arrows indicate the expected size of each protein. M) marker, A) a periplasmic sample of control strain, A ') supernatant sample of control strain, B) an intercellular space sample of OptA-expressing strain JOL1809, C) OptB E) Expression of intercellular space of JOL1812, E ') Expression of HIy in cell-space sample of JOL1810, C') OptB expression strain JOL1810, D) Intercellular space sample of FliC expression strain JOL1811, E) The supernatant sample of strain JOL1812.
FIG. 7 shows the results of confirming the activity of IgG against each antigen in a mouse group immunized with 4 kinds of JOL1800-derived strains expressing each Rosaceae antigen. Comparison of unimmunized mice (Group A, white bars) and mice immunized with all four antigens (Group J, gray bars). I) OptA-specific IgG, II) OptB-specific IgG, III) FliC-specific IgG, IV) Hly-specific IgG. * Indicates the largest significant difference between the control and immunized groups ( P <0.02).
FIG. 8 shows the results of confirming the activity of IgA against each antigen in a mouse group immunized with 4 kinds of JOL1800-derived strains expressing each Rosaceae antigen. FIG. 8A shows the vaginal mucosal sIgA (viginal sIgA), and FIG. 8B shows the activity of the intestinal sIgA (intestinal sIgA). Immunized mice (Group A, white bars), mice immunized to all four antigens (Group J, gray bars). V, IX) IgA, VII, XI specifically reacting with OptA, IgA, VII, XI specifically reacting with OptB, IgA, VIII, XII) specifically reacting with FliC, IgA. * Indicates the largest significant difference between the control and immunized groups ( P <0.02).
FIG. 9 shows the results of T cell immune responses of splenocytes isolated from mice using FACS. I) Comparison of T cell changes in CD3 ⁺ CD4 ⁺ T cells and CD3 ⁺ CD8 ⁺ in unimmunized and immunized mice. II) degree of CD3 ⁺ T cells, CD3 ⁺ CD4 ⁺ T cells and CD3 ⁺ CD8 ⁺ T cells increased in immunized mice for nonimmunized mice. Immunized mice (JOL1800) mixed with non-immunized mice (A) and four strains derived from JOL1800 expressing each Rosaceae antigen.
FIG. 10 shows the results of expression of a cytokine gene measured by re-stimulating splenocytes isolated from mice immunized with a mixture of non-immunized mice and four strains derived from JOL1800 expressing each antigenic antigen. The amount of cytokine expressed in unimmunized mouse group A was 1, indicating an increase in the relative expression level. Splenocytes from mice belonging to group J were divided into OptA, OptB, FliC, and Hly, respectively, to stimulate the expression of cytokine.

In order to accomplish 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 L. intracellularis (hereinafter also referred to as L. intracellularis ) is a gram-negative intracellular parasitic organism and is known as a causative organism of porcine proliferative pyelonephritis.

In order to select the Rho antigen of the present invention, LI0649 (802639-805194), which encodes the autotransporter protein, and flagellin, respectively, were synthesized on NCAI's rosinia intracellularis PHE / MN1-00 gene map The genomic sequence of LI0570 (701958-702842) coding for hook associated flagella C and LI0004 (3454-4209) gene coding for hemolysin were confirmed. To determine the vaccine antigen compatibility of each gene, the epitope of this antigen protein was determined using the ProteomeBinders Epitope Choice Resource program (http://bioware.ucd.ie/epic/). The antigenic determinant for B cell expression for the humoral immune response is directly related to the water-soluble antibody. Therefore, the ratio of the hydrophilicity residue considering this and the antigenicity considering the structure of the protein, A region having a high function as a crystallizer was predicted.

One of the Gram-negative bacterial outer membrane proteins, Autotransporter (Opt), is an N-terminal signal peptide, a passenger domain, a β-domain ), And the protein synthesized in the passenger domain serves to transport the N-terminal signal peptide to the cytoplasmic membrane, while the protein in which the β-domain is transported to the cytoplasmic membrane is called the outer membrane It functions to create a passage that can express. The pathogenicity of the passenger domain protein is known to play a role in the adhesion, invasion and bacterial toxicity of the target host or cells of the host. However, attempts have been made in the live vaccine to prevent PPE There is no enemy.

The OptA and OptB antigens comprise the first region predicted to be the most antigenic of the LI0649 gene (802639-805194) encoding the Rhodiola intracellularis-derived Autotransporter protein, a portion of the passenger domain The 101 to 200th amino acid sequence portion was selected as the first candidate protein (OptA), and the 534th to 851st amino acid sequence portion including a part of the passenger domain and the entire? -Domain was determined as the second candidate protein (OptB).

The scope of the OptA antigen according to the present invention includes proteins having the amino acid sequence shown in SEQ ID NO: 1 and functional equivalents thereof. Is at least 60% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, more preferably 90% or more, Refers to a protein having a homology of at least 95% and exhibiting substantially the same physiological activity as the protein represented by SEQ ID NO: 1. "Substantially homogenous physiological activity" means a porcine proliferative, retroviral, vaccine activity. The invention also encompasses fragments, derivatives and analogues of the OptA antigen.

The scope of the OptB antigen according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and functional equivalents thereof. The specific contents of the functional equivalents of the proteins are as described above. It also includes fragments, derivatives and analogues of OptB.

The FliC antigen was selected as a gene (Lawsonia PHE / MN gene map LI0570 gene) encoding a hook associated flagella C that synthesizes flagellin, a protein that constitutes flagella . The flagella of the cell surface that regulates bacterial movement usually acts as pathogen-associated molecular patterns (PAMPs) and is involved in the activation of the acquired immune system as a component of the innate immunity in the host (Toll like receptor 5, TLR5), which is one of the factors that stimulate NF-κB in the macrophage, contributing to the activation of the acquired immune system by activation of NF-κB. However, the plasgela gene has not yet been used as a candidate gene for the PPE live vaccine.

In this study, the protein associated with the flagella hook was selected as the antigen. The selected region of the antigen is a flagellin N-terminal helical portion and a C-terminal helical portion conserved in size of 885 nt (701958-702842) Lt; RTI ID = 0.0 &gt; PAMPs &lt; / RTI &gt;

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

The Hly antigen of the present invention was selected from the hemolysin (Hly) gene (LI0004 gene of the Lawsonia PHE / MN gene map) derived from the LA intracellularis. Rosonia intracellularis is free from the phagolysosomal fusion of the host and degrades the entry vacuole that forms during intrusion into the cytoplasm. Hemolysin is a membrane-damaging cytolytic enzyme And it is known to help decompose the incoming liquid. On the 5th day of infection, the cells were evolved from cytoplasm free from vacuoles in villus and crypt cells, and proliferation of ileum and cells was observed 10-12 days after infection.

The selected site of the Hly antigen is 756 nt (3454-4209) in size and synthesizes hemomysin A protein known as bacterial pore forming toxin. As a virulent factor, the hemorrhagic gene has been widely used as a candidate gene for the development of vaccines for other bacteria, but has not yet been used as a candidate gene for the PPE live vaccine.

The scope of Hly according to the present invention includes proteins having the amino acid sequence shown in SEQ ID NO: 4 and functional equivalents thereof. The specific contents of the functional equivalents of the proteins are as described above. It also includes fragments, derivatives and analogues of Hly.

The attenuated Salmonella mutant of the present invention may be one in which the asd gene has been deleted. Preferably, Salmonella mutants in which the lon, cpxR and asd genes are deleted, more preferably Salmonella mutants in which the lon, cpxR, rfaL and asd genes are deleted, are not limited thereto.

In Salmonella mutant strains in accordance with one embodiment of the invention, lon, cpxR and asd gene is a Salmonella mutant deleted (Δlon ΔcpxR Δasd Salmonella Typhimurium, JOL912 ) are antigen without antibiotics as DAP (diaminopimellic acid) which the asd gene deletion request weeks Not only is it made to select recombinant strains, it can increase lymphocyte infiltration ability by deletion of cpxR , increase immunogenicity, lose lon gene and attenuate pathogenicity. It is known that the strain has no effect on the production of EPS (extracellular polysaccharide) which can act as an antigen, and thus acts as an antigen itself, resulting in sufficient humoral mucosal cellular immune response with stability.

In one embodiment of the present invention, the deletion of genes related to the expression of Lipopolysaccharide (LPS) of Salmonella was carried out in order to increase the expression of external antigens in a conventional S-type smoothed Salmonella strain (JOL912) . LPS is a part of the outer membrane of Gram-negative bacteria, which removes the rfaL gene involved in the synthesis of the O-antigen polysaccharide (O-PS) polymerase of LPS and determines the length of LPS synthesized on the outer membrane Shortened. The thus developed O-antigen-free rough Salmonella mutant ( Δlon ΔcpxR Δasd ΔrfaL, JOL1800) retains the antigenicity of LPS and inhibits the expression of LPS in the extracellular membrane expression of the cellulase intracellularis antigen gene selected on the extracellular membrane By being exposed to the host's immune system without being screened, the immune response stimulated by external antigens could be enhanced.

According to one embodiment of the present invention, JOL1800 survived in the host's spleen to such an extent that it effectively penetrates macrophages in mice and induces an immune response regardless of the route of inoculation. However, the serum complement was more easily killed than the wild-strain-derived attenuated Salmonella strain (JOL912). The attenuated JOL1800 with O-antigen removed is more sensitive to serum complement, but it is supposed to compensate for weakened defense mechanism by avoiding the LPS-specific antibody of the individual that has already been exposed to Salmonella strains.

An additional benefit of using Salmonella strains with the O-antigen removed as a delivery vector is DIVA (Differentiation of Infected and Vaccinated Animals). When JOL1800, a strain prepared by completely removing the O-antigen, which is the outermost constituent repeating sugar polymer of LPS, is used as a vector for transferring heterologous antigen, the serum ELISA at the time of vaccination more than 4 weeks, It can be used for DIVA which distinguishes individuals immunized with infected and O-antigen-deficient vaccine strains.

The attenuated Salmonella mutant of the present invention may be any one selected from the group consisting of a Bla (beta-lactamase) signal sequence, a gene encoding the OptA, OptB, FliC and Hly antigens linked thereto; And an asd gene; and the like. However, the present invention 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) with 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 yphimurium t), Salmonella tie blood (Salmonella typi), Salmonella para Thai Phi (Salmonella paratyphi ), Salmonella ( Salmonella sendai ), Salmonella ( Salmonella gallinarium) or Salmonella Entebbe utility disk (Salmonella enteritidis ), and the like, preferably Salmonella typhimurium, but is not limited thereto.

In addition,

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

(d) selecting the transformed Salmonella mutant strain of step (c). The present invention also provides a method of producing an attenuated salmonella mutant for the simultaneous prevention or treatment of swine growth regulatitis and salmonellosis.

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

The Salmonella mutant mixture of the present invention is characterized in that the Salmonella mutant in which the lon, cpxR, rfaL and asd genes have been deleted is selected from the group consisting of Salmonella mutant strains expressing either an antigen selected from the group consisting of OptA, OptB, FliC and Hly antigens, Or a mixture thereof.

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

In addition, the present invention provides a vaccine composition for preventing or treating swine proliferative ileitis and salmonellosis which comprises the attenuated salmonella mutant or a mixture thereof as an active ingredient.

The vaccine composition of the present invention contains Salmonella mutants attenuated by gene deletion as an active ingredient in a mixture containing at least one Salmonella mutant expressing OptA, OptB, FliC and Hly antigens derived from Lawsonia intracellularis , The vaccine composition can be administered to a human or an animal to prevent or treat swine proliferative ileitis and salmonellosis at the same time.

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

The composition of the present invention may be administered orally or parenterally (for example, intramuscularly, intravenously, subcutaneously, intraperitoneally or topically), preferably orally or subcutaneously, depending on the intended method , But is not limited thereto. The dosage of the composition may vary depending on the weight and age of the human being, the sex, the health condition, the diet, the administration time, the administration method, the excretion rate, and the severity of the disease.

The vaccine composition may be inoculated into a human or mammal, and the mammal may be a cow, a deer, a goat, a goat, a dog, a pig, and the like, preferably, but not limited to, a pig.

The term "vaccine " in the present invention refers to a biological agent containing an antigen that immunizes a living body. The term &quot; vaccine &quot; refers to an immunogenic or antigenic substance that causes immunization to a living body by injection or oral administration to a human or animal for the prevention of infection. In vivo immunity is largely divided into autoimmunity, which is obtained automatically in vivo after infection with pathogenic bacteria, and passive immunization, which is obtained by externally injected vaccine. While autoimmunity has a long period of immune-related antibody production and is characterized by persistent immunity, passive immunization by vaccine acts immediately for the treatment of infectious diseases but has a disadvantage that its persistence is poor.

The vaccine composition may contain one or more of a stabilizer, an emulsifier, aluminum hydroxide, an aluminum phosphate, a pH adjuster, a surfactant, a liposome, an iscom adjuvant, a synthetic glycopeptide, an extender, a carboxy polymethylene, a subviral particle adjuvant, , N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, dimethyl dioctadecyl- ammonium bromide, The second auxiliary agent may be further contained.

In addition, the vaccine composition may comprise a veterinarily acceptable carrier. The term "veterinarily acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, Examples of the carrier, excipient and diluent which can be contained in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, each of the above-mentioned compositions for the vaccine may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, and nasal formulations such as drips or sprays, And the like. In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. As the liquid preparation for oral administration, suspensions, solutions, emulsions, syrups and the like may be used. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, and freeze-drying agents. Examples of the suspending agent include propylene glycol, polyethyleneglycol, vegetable oil such as olive oil, injectable ester such as ethylolate, and the like, but the present invention is not limited thereto. Penetrants suitable for formulation for intranasal administration are generally known to those skilled in the art. Such suitable formulations are preferably formulated to be sterile, emollient and buffered for stability and compliance. Formulations for intranasal administration are also prepared to stimulate mucus secretion in several ways to maintain normal ciliary action and are described in Remington's Pharmaceutical Science, 18th Ed., Mack Publishing Co., Easton, PA (1990) As noted, suitable formulations are preferably isotonic, slightly buffered formulations that maintain a pH of 5.5 to 6.5, and most preferably comprise an antimicrobial preservative and a suitable drug stabilizing agent.

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

The feed additive of the present invention comprises Salmonella mutants expressing and secreting OptA, OptB, FliC and Hly antigens derived from Lawsonia intracellularis , or a mixture thereof as an active ingredient, wherein the Salmonella mutant or a mixture thereof is Rhodiola And salmonella as well as the cellular and humoral immune response of the animal. Therefore, when it is used as a feed additive, it can contribute to prevention of simultaneous proliferation of pork and salmonellosis of domestic livestock and immunity enhancement.

The feed additive of the present invention can be prepared by using the Salmonella mutant mixture as it is or adding a known carrier such as cereal grains and by-products, stabilizers and the like which are allowed to be added to livestock. If necessary, citric acid, fumaric acid, adipic acid, Citric acid, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, and citric acid, and organic acids such as citric acid and malic acid, sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate Natural antioxidants such as tannic acid and phytic acid, antibiotics, antimicrobial agents and other additives may be added, and the shape thereof may be a proper state such as powder, granule, pellet, suspension, etc. In the case of feeding the feed additive, And the like can be supplied alone or in a mixed form to the feed.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Development of O-antigen deficient Salmonella strains

1.1. The strains and plasmids used in the experiments

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

1.2. Experimental animal

All laboratory animal care and procedures described in this invention were approved by the Animal Ethics Committee of Chonbuk National University (CBNU2015-00085) in accordance with the guidelines of the Korean Committee on Animal Care. Five - week - old BALB / c female mice were purchased and housed in laboratory animal husbandry at Chonbuk National University.

1.3. Production of O-antigen deletion mutants

We have developed strains that lack O-antigen in LPS of bacterial surface by removing o-antigen ligase gene rfaL from existing attenuated S. Typhimurium. Plasmid pKD46 containing lambda red, which induces recombination, was transformed into an attenuated strain S. typhimurium JOL912 ( ΔcpxR, Δlon, Δasd ) by electric shock. After culturing at 32 ° C or lower (28-30 ° C), it was confirmed whether the plasmid was successfully introduced by PCR. The pKD3 plasmid was used as a template and amplified by PCR using a primer having a length of 40 nucleotides including the rfaL gene and a part of the pKD3 plasmid. The amplified PCR products were extracted and transformed into S. typhimurium competent cells into which pKD46 was introduced. The cells were plated on LB (Luria-Bertani) solid medium supplemented with Arabinose and cultured at 37 ° C. The recombinant strain was selected on LB solid medium containing 25 占 퐂 / ml chloramphenicol. Knock-out was confirmed by PCR using primers. To remove the antibiotic resistance gene, a competent cell was constructed from colonies identified as transgenic to transform the pCP20 plasmid. LB medium, and cultured at 37 DEG C to select a bacterium having no 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. In vitro complement sensitivity assay

Serum complement sensitivity of S. Typhimurium was measured using rabbit serum. Serum without anti-Salmonella antibody was collected from rabbits and used for analysis. All strains used were cultured to the late log phase and diluted to 1 x 10 ⁷ cfu / 100 μl. Each of the prepared JOL401, JOL912, and JOL1800 cultures was mixed with 100 占 퐇 of PBS, 100 占 퐇 of 50% serum complement, and 100 占 퐇 of complement-free serum (DCS), followed by incubation at 37 占 폚 for 1 hour. After that, all cultures were plated on 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. Macrophage phagocytosis / penetration analysis

The primary peritoneal macrophage of mice was infected with JOL401, JOL912, and JOL1800, and the phagocytosis and infiltration of the bacteria were observed using a fluorescence microscope. Macrophages were collected by injecting 5% BSA into the abdominal cavity of 4-week-old mice. Macrophages were treated aseptically and dispensed onto coverglass slips coated with 0.2% gelatin at 5 × 10 ⁶ cells. Then, RPMI [RPMI 1640, 10% FBS (heat-inactivated fetal bovine serum), 1 x Antibiotic-Antimycotic (Gibco, Life technologies, USA)] was poured into 6-well cell culture plates. After incubation in an incubator at 37 ° C and 5% CO ₂ for 24 hours, macrophages were infected with S. Typhimurium at an MOI of 10: 1. After the bacterial treatment, the cells were cultured in a 5% CO ₂ incubator at 37 ° C for 30 minutes. Bacteria that were not digested or infiltrated were washed with PBS, added with RPMI containing antibiotics, and placed in an incubator for another 48 hours. Cells from the cover slips were fixed with 3% paraformaldehyde diluted in PBS and immunofluorescent stained. The primary antibodies were chicken anti-912 polyclonal sera when infected with JOL401 and JOL912 and chicken anti-1800 polyclonal sera when infected with JOL1800. Were diluted 1: 1,000 in 0.1% tween 20 PBS. All secondary antibodies were diluted in PBS at a ratio of 1: 5,000 to goat anti-chicken IgY H & L-Alexa Fluor 488 (Abcam, UK). The nuclei of macrophages were counterstained with 0.5 μg / ml of DAPI (2- (4-amidinophenyl) -1H-indole-6-carboxamidine; Sigma-Aldrich, US). The cover slip was attached to a micro-slide and observed with a fluorescence microscope (AX1 Zeiss., Germany). Macrophages were observed 4, 24, and 48 hours after infection, respectively.

1.6. Survival analysis of Salmonella strains

To determine how long the S. Typhimurium strain of the present invention remains in the substantial organs of the host. A total of 96 rats were divided into 3 groups (n = 32). JOL401 and JOL1800 were inoculated at 1 × 10 ⁷ cfu into 4-week-old mice respectively, and JOL1800 was inoculated intramuscularly into the third group. For the purpose of the experiment, JL1800 asasd strain was transformed with asd + plasmid pJHL65 by electric shock. Eight mice of each group were anesthetized at 3, 7, 14, 21, and 28 days after inoculation and aseptically harvested the spleen. To confirm the presence of Salmonella in the spleen, the spleen was homogenized with 2 ml of BPW (buffered peptone water, Becton, MD, USA) using a homogenizer. 100 占 퐇 of the homogenized solution was immediately inoculated evenly onto a BGA (Brilliant Green Agar) plate and cultured overnight at 37 占 폚. At the same time, the remaining BPW samples were inoculated on RV (Rappaport-Vassiliadis) medium and cultured at 37 DEG C for 48 hours. The resulting colonies were finally confirmed by PCR using S. typhimurium specific primers.

1.7. Identification of DIVA ability based on serum IgG

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

2. L. intracellularis  Development of vaccine strains expressing antigens

2.1. Strains and plasmids

The bacterial strain and plasmid used in the present invention can be obtained by It is listed in Table 1. Since the pET28a and pET32a plasmids, which are commercially available E. coli protein expression vectors, have kanamycin resistance, the strain was cultured in the presence of kanamycin (50 μg / ml). The Bl21 (DE3) pLysS strain was used for the purification of the LosA antigen protein. The strain was cultured in the presence of 0.1 M IPTG (Isopropyl beta-D-1-thiogalactopyranoside) to induce over-expression in protein purification. The pJHL65, pJHL80 asd + plasmid was used as a vector to secrete and transfer cloned antigen proteins in a periplasmic secretion manner between Bla signal sequence-based cells. All strains were cultured at 37 ° C using LB medium and stored at -80 ° C using LB liquid medium containing 20% glycerol. DAP was added when culturing JL1800 asd- strain at a concentration of 50 占 퐂 / ml.

2.2. Production of plasmid expressing antigen protein

The candidate antigens of L. intracellularis are OptA and OptB related to auto-transporter synthesis, FliC to synthesize the flagella-related protein, and Hly, which is thought to be a toxin related to cell infiltration. All genes necessary for expression of antigen protein were obtained using gene synthesis (Bioneer, Korea). Genes of L. intracellularis that synthesize each protein were examined by GenBank of National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). Each L. intracellularis antigen was analyzed using the Peptide Property Calculator software (http://www.biosyn.com/peptidepropertycalculator/), and the nucleotide sequences of highly antigenic sites were selected and synthesized. The genes were cloned into the pBHA vector by adding the restriction enzyme sites on both ends. The restriction enzyme sites added at both ends of the L. intracellularis antigen gene are Sal I and Xho I OptA, Sal I and Pst I OptB, EcoR I and Sal I FliC, and EcoR I and Hind III Hly, respectively. The desired restriction endonuclease was used to obtain the desired antigen DNA fragment, and the recombinant plasmids pET32a-OptA, pET28a-OptB, pET28a-FliC and pET28a-Hly were prepared by cloning into plasmids pET28a and pET32a, respectively. OptA was not inserted into pET28a and was cloned into pET32a, a similar line of plasmids. The obtained recombinant plasmid was transformed into E. coli BL21 (DE3) pLysS by thermal shock method to prepare a strain for protein purification that overexpressed the antigen protein in the presence of IPTG. The presence or absence of the L. intracellularis antigen gene in the strain was confirmed by electrophoresis and PCR performed on agarose gel after digesting the plasmid from BL21 (DE3) pLysS into restriction enzyme corresponding to each antigen. The primers used for PCR amplification were as 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), and JOL1742 (BL21 containing pET28a and expressing Hly). In each strain, OptA, OptB, FliC, and Hly proteins were purified using NTA (Ni-nitrilotriacetic acid) agarose (Qiagen, Valencia, Calif.). The antigen protein of L. intracellularis obtained by culturing and purifying each strain was used to detect specific antibodies against each antigen in mice.

To construct the final target strains L. intracellularis and S. Typhimurium co-prophylactic vaccine candidates, pJHL65-OptA, pJHL65-OptB, pJHL65-FliC, and pJHL65-OptA were constructed by cloning the antigen gene into the pJHL65 and pJHL80 plasmids, , pJHL65-Hly.

2.3. Production of vaccine strain

PJHL80-OptA, pJHL65-OptB, pJHL65-FliC, and pJHL65-Hly prepared in 2.2 were transformed into JOL1800 by electric shock. JOL1800 was washed twice with distilled water containing 10% glycerol, mixed with 0.1 μg of the purified plasmid in a cuvette, and then subjected to electric shock with Bio-Rad MicroPulser (Bio-Rad, USA) 0.0 &gt; 37 C &lt; / RTI &gt; for 1 hour. 100 [mu] l of the cultured mycelia to be screened for transformed Salmonella strains were plated on LB agar without DAP, and then dried and cultured overnight, and colonies formed were selected. The plasmid was recovered from the selected strains, digested with restriction enzymes corresponding to each antigen, and confirmed by electrophoresis and PCR performed on agarose gel.

2.4. Western Blot

The antigens of L. intracellularis expressed in the outer membrane of the prepared vaccine strain and in the surrounding cytoplasmic space were identified by Western blotting, respectively. OptA in JOL1809, OptB in JOL1810, FliC in JOL1811, and Hly in JOL1812 were expressed. Antigen proteins expressed in the recombinant strains were obtained from cell - free supernatants through TCA precipitation. When the optical density (OD ₆₀₀ ) was measured at ₆₀₀ nm in LB liquid medium at 37 ° C. in order to prepare the antigen protein secreted by the recombinant plasmid having the recombinant plasmid, the bacteria were cultured until 0.8 was obtained and centrifuged at 3,400 × g for 20 minutes The supernatant was separated. A 20% (v / v) trichloroacetic acid (TCA) solution was added to the clear supernatant obtained by passing the supernatant through a 0.22 μm filter and reacted overnight at 4 ° C. The supernatant was centrifuged at 15,700 × g for 30 minutes, and the precipitated pellet was washed with acetone and resuspended in PBS. The resulting solution was mixed 1: 1 with SDS-PAGE sample buffer and used. For comparison, the attenuated Salmonella strains with pJHL65 vector were used in the same manner as the control group. Each protein sample was separated by SDS-PAGE for Western blot analysis and transferred to 0.2 μm PVDF membrane (Millipore, Billerica, MA, USA) and blocked with blocking buffer (3% BSA, PBS, 0.1% Tween- The reaction was allowed to proceed overnight. The following day, an anti-his-tag antibody (IG Therapy Co., Ltd., Korea) was diluted 1: 5,000, reacted for 1 hour, and HRP (horseradish peroxidase) -conjugated goat anti-mouse IgG diluted 1: 5,000 Time. The results were stained using 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. L. intracellularis Antigen synthesis experiment

3.1. Strains and plasmids

The strains and plasmids used in this experiment were JOL1809, JOL1810, JOL1811, and JOL1812, recombinant attenuated Salmonella strains expressing the L. intracellularis antigen shown in Table 1.

3.2. Experimental animal

L. intracellularis Sixteen mice were divided into two groups and used for the experiment (n = 8) for the immunogenicity of OptA, OptB, FliC and Hly antigen. The following conditions are the same as 1.2.

3.3. Vaccine preparation

JL1809, JOL1810, JOL1811, and JOL1812, O-antigen deficient Salmonella strains expressing L. intracellularis antigens OptA, OptB, FliC, and Hly, respectively, were grown in LB solid medium, and five colonies were inoculated into LB liquid medium Lt; 0 &gt; C overnight. The separately cultured strains were added to LB liquid medium to be diluted at a ratio of 1: 100, respectively, and cultured for 3 to 5 hours until an OD ₆₀₀ value reached 0.6. The culture was centrifuged at 190 × g at room temperature and the precipitate was washed three times with sterile PBS. The final precipitate was suspended again in sterile PBS, and the OD ₆₀₀ value was determined to determine the number of bacteria. JOL1809, JOL1810, JOL1811, and JOL1812 were mixed at a concentration of 5 × 10 ⁷ CFU to determine the final inoculum amount of 2 × 10 ⁸ CFU / 100 μl.

3.4. vaccination

Inoculation of the vaccine was carried out in order to confirm the induction of the immune response when inoculated with four antigens of L. intracellularis OptA, OptB, FliC and Hly. The mice were divided into two groups (n = 8). One group was subcutaneously inoculated with PBS sterilized as a control group, and the other group was mixed with 5 x 10 ⁷ CFU each of JOL1809, JOL1810, JOL1811 and JOL1812, 10 &lt; ⁸ &gt; CFU / 100 [mu] l of the strain was subcutaneously inoculated.

3.5. Sampling

Blood samples were collected for vaginal, intestinal, and IgG measurements for sIgA before and 2 weeks after inoculation. For vaginal lavage, the vaginal washes were performed using PBS and the solution was stored at -20 ° C. In the case of intestinal lavage fluid, pilocarpine-based lavage was performed. Wash liquid (4 ml of Lavage base, 6.5 g of Poly ethylene glycol, DW 40 ml) is prepared, and 500 μl of the solution is fed to the probe. 20 minutes later, 100 μl of pilocarpine 0.5% is injected per abdominal cavity. Each of the feces collected in the plate was treated with 500 μl each of 50 mM EDTA and centrifuged at 3,400 × g for 15 minutes. The supernatant was separated and stored at -20 ° C. Serum was collected from the orbital vein and centrifuged at 4000 × g for 5 minutes. The supernatant serum was separated and stored at -20 ° C.

3.6. ELISA analysis

ELISA was performed to determine the specific IgG and IgG levels of L. intracellularis OptA, OptB, FliC and Hly antigens expressed in recombinant vaccine strains. OptA, OptB, FliC and Hly antigen proteins purified from strains JOL1586, JOL1593, JOL1682 and JOL1742 were used as coating antigens. The standard protein wells were coated with the purified antigen protein at a concentration of 500 ng / well and the chlorine-anti-mouse IgG or goat anti-mouse sIgA at a concentration of 200 ng / well, respectively, followed by coating overnight at 4 ° C. The coated plates were washed 3 times with PBS (PBST) containing 0.05% Tween 20 and blocked with blocking buffer (3% skim milk in PBS) for 1 hour at 37 ° C. Serum and PBS were diluted 1: 100, diluted 1: 3 with vaginal washing solution and 1: 4 with intestinal wash solution, and dispensed into wells at 100 each, followed by reaction at 37 캜 for 1 hour. Mouse anti-mouse IgG HRP for serum and goat anti-mouse IgA HRP for vaginal washes were diluted at a ratio of 1: 5,000, and 100 μl each was dispensed into each well, followed by reaction at 37 ° C for 1 hour. The OPD-substrate reaction solution was dispensed in 100 쨉 l / well, color-developed, stopped with 3M H ₂ SO ₄ , and the OD value was measured at 492 nm. The concentration of each antigen-specific antibody was measured based on standard protein concentration.

3.7. T cell immune response measurement

The control and immunized mice were each grouped (n = 5). After 10 days of vaccination, the mice were sacrificed and the aspirated spleen was collected. The mice were then pulverized, the tissues were removed from the tissues, and the remaining tissue was removed with a cell strainer Respectively. After centrifugation at 470 x g for 3 minutes, the pellet was suspended with RPMI 1640 and RBC was removed by floating the cells using RBC elution buffer. And washed twice with RPMI 1640, and the final pellet was suspended with RPMI. The number of cells suspended in RPMI was measured and dispensed into 96-well plates at 1 x 10 ⁶ cells / well. Mouse anti-mouse CD3e-PE, anti-mouse CD4-perCP-vio700 and anti-mouse CD8a-FITC were reacted for 15 min at 4 ° C in dark conditions. The stained cells were washed three times with 200 μl FACS buffer and analyzed using a MACSQuant® analyzer (miltenyi Biotec, Germany).

3.8. Measurement of cytokine mRNA by reverse transcription real-time PCR

Cells were dispensed into 96-well plates in the same manner as in 3.7. OptA, OptB, FliC, and Hly were stimulated with 200 ng / ㎕ in 1 × 10 ⁶ cells for 48 hours under 5% CO ₂ environment condition. After incubation, total RNA was extracted using GeneAll® Hybrid-RTM kit and synthesized with cDNA using ReverTra Ace® qPCR RT Kit. The primers for mouse interferon-gamma (IFN-y) and interleukin (IL) -4, IL-17 used in real-time PCR are listed in Table 3. Real-time PCR was performed using SYBR Green Real-Time PCR Master Mix (QPK-201, TOYOBO, Japan). Were measured using a Step One plus Real Time PCR system (Applied Biosystems). For each cytokine, the amount of RT-PCR product was normalized to the beta -actin value using as an internal standard.

In the present invention, primers used for RT-PCR antigen Primer direction Sequences (5 '-> 3') SEQ ID NO: IFN-y
Forward TCA AGT GGC ATA GAT GTG GAA GAA 13 Reverse TGG CTC TGC AGG ATT TTC ATG 14 IL-4
Forward ACA GGA GAA GGG ACG CCA T 15 Reverse GAA GCC CTA CAG ACG AGC TCA 16 IL-17
Forward ACC GCA ATG AAG AT ACC CTG 17 Reverse TCC CTC CGC ATT GAC ACA 18

3.9. Statistical analysis

STATA (Statata Statistical Software, Release 8.0, Stata Corporation, College Station, TX) was used for statistical analysis. Antibody titer data from ELISA, CD3 +, CD4 +, CD8 + T cells, and fold change (2- ^ΔΔCT ) were considered significant when P <0.05. Unless otherwise specified, it is expressed in standard deviation (SD).

Example 1. O-antigen deficiency attenuation S.  Typhimurium strain development

1.1. Production of Salmonella live vaccine vector

The DIVA-capable S. Typhimurium live vaccine vector, JOL1800, was constructed by modifying JOL912, an attenuated S. typhimurium strain with high immunogenicity and permeability. JOL912 is an auxotrophic mutant strain that deletes three genes ( Δlon ΔcpxR Δasd ), and DAP is essential for replication due to deletion of the asd gene. Thus, the asd + plasmid is used as a vector expressing an antigen gene to satisfy balanced-lethal complementation. Lamma by the deletion of additional rfaL O- antigen ligase gene using a Red recombination JOL912 prepare a new Salmonella vaccine vector, Δ lon Δ cpxR Δ Δ asd rfaL JOL1800 (Fig. 1). PKD46, a lambda RED plasmid, was transformed into JOL912 by electroporation and a cat gene cassette amplified by PCR in pKD46 transformed strain was introduced. The bacteriophage recombination system, called gamma-red, has the gamma, beta, and exo genes. Exo and Bet genes work together to promote gene recombination. Exo acts on both ends of ds DNA to make a 3-overhang and bet binds to 3 ss DNA and causes strand exchange by RecA. The cat gene cassette exchanged here made the plasmid pKD3 as a template. When pKD46 and JOL912 transfected with linear DNA were cultured at 37 ° C, recombination occurred and rfaL was replaced with a cat gene cassette. At the same time, pKD46 exits the cell. The removal of the target gene was confirmed by PCR using a primer of the rfaL flanking region. When wild rfaL is converted to the cat gene cassette (~ 1.1kb), the size of the entire 1.9kb amplicon is reduced to 1.7kb. Plasmid pCP20 was transformed into the strain in which the removal of the rfaL gene was confirmed. pCP20 was introduced to remove the antibiotic resistance gene and expressed FLP recombinase directly acting on the FRT (FLP recognition target) site at both ends of the antibiotic resistance gene cassette. Thereafter, the strain was cultured at 30 ° C to obtain a strain in which the cat gene cassette was removed, and further cultured at 37 ° C or 40 ° C to obtain a final strain free of the plasmid pCP20. The last isolate was named JOL1800, a mutant strain lacking the antibiotic resistant lon, cpxR, asd, and rfaL genes derived from JOL912. JOL1800 is a R. salmonella strain with no O-antigen due to the removal of the rfaL gene. Separation of PAGE from JOL1800 After the purified LPS was extracted, it was confirmed that the O-antigen on the surface of the microorganism was not present by silver staining (FIG. 2). In the smooth JOL912 used as a control, long-expressed O-antigen can be seen.

1.2. In-bit complement sensitivity analysis

To investigate the effect of O-antigen length on complement dependent cytotoxic effects, wild-type S. typhimurium JOL401, attenuated strain JOL912, and JOL1800, an O-antigen-deficient strain derived from JOL912, were cultured to conduct complement sensitivity analysis. R-type JOL1800 showed a more sensitive response to rabbit complement than the other S-type strains. JOL1800 showed a significant decrease in the number of bacteria compared to wild JOL401 and JOL912 (FIG. 3).

1.3. Macrophage phagocytosis / penetration analysis

The mouse macrophages were infected with wild-type S. Typhimurium JOL401, an attenuated S. Typhimurium JOL912, and an O-antigen-deficient S. Typhimurium JOL1800 derived from JOL912, thereby confirming the difference in the ability of the three strains to phagocytose or infiltrate macrophages .

As a result, JOL401, JOL912, and JOL1800 were able to confirm infiltration of bacteria into macrophages 4 hours after infection. Salmonella has an infection pathway in which bacteria live in the host macrophages and spread throughout the body. In addition to JOL912, which was attenuated by removing the lon, cpxR, and asd genes , JOL1800, in which the rfaL gene was deleted and the O-antigen of the bacterial surface LPS was additionally removed, was also confirmed to have ability to infiltrate into macrophages and to survive ).

1.4. Survival analysis of Salmonella strains

Mutant strains produced by deletion of several genes are highly attenuated and sometimes fail to reach the actual organs or die easily and sometimes do not cause infection. When an attenuated strain is used as a vaccine, appropriate attenuation control is necessary to prevent the vaccine strain from surviving too long in the host to cause chronicization of the disease or to survive too short to cause an immune response. The mice were infected with JOL401, JOL912, and JOL1800, and the spleen was collected after 3 days, 7 days, 14 days, and 21 days to check for the presence of bacteria (Table 4). All strains were detected in the spleen at 3, 7, and 14 days after inoculation. JOL401 and JOL912 survived in the spleen 21 days after inoculation. The survival time (recovery time 50) of JOL1800 was investigated through PROBIT analysis for 12 days. It can be seen here that JOL1800 penetrates into the host's immune system and induces a sufficient immune response.

1.5. DIVA based on serum IgG

To investigate the possibility of DIVA of the developed strain JOL1800, ELISA was performed on sera from mice inoculated with LPS extracted from S. Typhimurium. The results obtained after inoculation with JOL401, JOL912, and JOL1800 measured at OD ₄₉₂ were compared. If there is a significant difference in the measured values, it can be used for DIVA. JL401 and JOL912, which are O-antigen-presenting strains, showed LPS-specific antibodies whereas JL1800, an O-antigen-depleted R-type strain, did not detect LPS-specific antibodies (FIG. Boosting was performed 3 weeks after the first inoculation in consideration of the inability to induce LPS - specific antibodies due to rapid in vivo elimination reaction due to attenuation of JOL1800. However, LPS-specific antibody response did not appear after that, and it is considered that DIVA is possible when JOL1800 is used as a vaccine. After 4 weeks of inoculation, ELISA was performed on the serum of each individual, and the difference in absorbance measured at OD ₄₉₂ revealed that only natural and O-antigen-deficient vaccine strains exposed to S. Typhimurium with O-antigen were exposed It is possible to distinguish the individual objects.

Example 2. L. intracellularis  Development of vaccine strains expressing antigens

2.1. Vaccine production and development

The plasmids pJHL80-OptA, pJHL65-OptB, pJHL65-FliC and pJHL65-Hly prepared by cloning the synthesized L. intracellularis antigens OptA, OptB, FliC and Hly into the antigen expression plasmids pJHL65 and pJHL80, The vaccine strain was transformed by electrophoresis into JOL1800, an attenuated S. Typhimurium strain capable of DIVA as an antigen. pJHL65 and pJHL80 express the cloned antigen in the intercellular space or surface of the bacteria. Then, the transformed plasmid was re-extracted, and the transformed plasmid was cut with the corresponding restriction enzymes and subjected to electrophoresis and PCR using agarose gel. The strains finally confirmed to have the antigen gene were named JOL1809 (OptA expression JOL1800), JOL1810 (OptB expression JOL1800 ), JOL1811 (FliC expression JOL1800), and JOL1812 (Hly expression JOL1800).

2.2. The presence or absence of antigen in the developed strain

JOL1809, JOL1810, JOL1811, and JOL1812 were subjected to Western blotting to confirm the presence or absence of each antigen. JOL1800 was used as a control group to compare antigen expression sites. The expression level of L. intracellularis antigen OptA was 17 kDa, OptB expression level was 41.5 kDa, FliC expression level was 38.6 kDa, and Hly expression level was 30 kDa (Fig. 6).

Example 3. L. intracellularis Antigen synthesis experiment

3.1. Immune response in whole body and mucosa

In order to evaluate the humoral immune response to each antigen when inoculated with the L. intracellularis antigens OptA, OptB, FliC, and Hly expressing JOL1800-derived S. Typhimurium strains, the antibody titers of IgG and sIgA in the rats were measured by ELISA Respectively. Serum IgG, vaginal mucosal sIgA, and intestinal mucosal IgA all showed antibody titers after the inoculation, elevated to about 4 weeks, and peaked and then decreased (Fig. 7 and Fig. 8).

3.2. T cell immune response

In order to evaluate the cellular immune response after inoculation with the recombinant attenuated S. Typhimurium live vaccines JOL1809, JOL1810, JOL1811, and JOL1812, FACS was used in splenocytes isolated from all immunized mouse groups and non-immunized control mice to generate T lymphocyte subgroup Respectively. All T cells are represented by CD3 ⁺ , while CD4 ⁺ and CD8 ⁺ are T helper cells and cytotoxic T cells, respectively. CD3 ⁺ CD4 ⁺ and CD3 ⁺ CD8 ^{+ as} well as the overall increase in CD3 ⁺ after 9 days of inoculation (Fig. 9). CD3 ⁺ T cells showed more than 50% increase, CD4 ⁺ was more than 45%, and CD8 ⁺ was more than 55% more than the control.

3.3. Measurement of cytokine mRNA by reverse transcription real-time PCR

RT-PCR was performed to measure the expression levels of IFN-y, IL-4 and IL-17 in order to evaluate the expression level of cytokines that regulate the cellular immune response. As a result of cytokine stimulation by splenocytes stimulated with spleen cells of mice subcutaneously inoculated with each strain expressing one Rosacea antigen, it was confirmed that IL-4 was significantly increased in OptA (FIG. 10) . All of the antigens except OptB showed a significant increase in IL-4 and IFN-γ. IL-17 is a cytokine secreted by differentiated Th17 cells, which stimulates secretion of all antigens. This suggests that CD4 ⁺ T cells are differentiated into Th1 and Th2 cells and that Th17 cells are differentiated.

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Vaccine composition for preventing or treating porcine          proliferative enteritis and salmonellosis simultaneouly          comprising attenuated Salmonella mutant as effective component <130> PN16501 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 100 <212> PRT <213> Lawsonia intracellularis <400> 1 Pro Ala Asn Ile Asn Gly Asp Ile Val Leu Ile Val Glu Asn Thr Asn   1 5 10 15 Thr Gln Asn Ser Ile Ile Gly Gly Ser Met Ala Asn Ala Ala Pro Val              20 25 30 Thr Ile Gly Gly Ser Ile Phe Met Thr Leu Arg Asn Val Thr Ala Val          35 40 45 Asp Pro Ile Phe Gly Gly Ser Val Asp Val Arg Phe Phe Ala Gln Gln      50 55 60 Gln Pro Asn Glu Asp Gln Leu Val Gly Gly Asp Ile Asn Ile Asn Leu  65 70 75 80 Glu Asn Val Thr Thr Pro Glu Phe Tyr Gly Leu Gly Tyr Ala Asn Gly                  85 90 95 Val Ile Pro Val             100 <210> 2 <211> 318 <212> PRT <213> Lawsonia intracellularis <400> 2 Ile Phe Asn Pro Gln Asp Lys Thr Trp Tyr Leu Thr Asn Phe Arg Gly   1 5 10 15 Ser Glu Asp Phe Tyr Gly Leu Ser Ala Ala Arg Glu Ala Ser Asn Trp              20 25 30 Leu Arg Gln Gln His Ile Trp Ser Leu Gln Arg Arg Ser Asn Lys Leu          35 40 45 Leu Asp His Gly Val Asp Gly Leu Trp Met Asn Val Gln Gly Gly Tyr      50 55 60 Glu Lys Leu Asp Ala Ala Ile Gly Asp Ala Lys Met Pro Trp Ile Met  65 70 75 80 Ala Ser Leu Gly Tyr Asp Phe Met His Lys Leu Ser Asp Phe Tyr Asn                  85 90 95 Leu Lys Ala Leu Tyr Gly Ply Gly Phe Gly Phe Ala Thr Gly Lys Asn             100 105 110 Lys Trp Asn Thr Ile Asn Ser Thr Asn Asp Ile Tyr Met Gly Leu         115 120 125 Val Gly Ala Tyr Val Gly Leu Met His Glu Ala Thr Gly Leu Tyr Gly     130 135 140 Thr Val Ser Gly Gln Phe Ala Thr Asn Arg Thr Lys Thr Lys Cys Thr 145 150 155 160 Gly Phe Asp Glu Thr Tyr Asn Trp Lys Glu Asn Val Pro Thr Glu Ala                 165 170 175 Ile Glu Ile Gly Trp Lys Trp Ser Ile Asp Glu Phe Lys Ile Asn Pro             180 185 190 Arg Gly Gln Val Ile Phe Glu Gln Leu Ser Lys His His Phe Ser Leu         195 200 205 Ser Gln Glu Gly Asp Thr Ala Ile Leu Asp Lys Glu Phe Leu Thr Thr     210 215 220 Thr Val Ile Gly Ile Ser Gly Glu Tyr Asp Leu Asp Leu Arg Ser Lys 225 230 235 240 Ile Ile Lys Leu Gln Ala Ser Val Asp Trp Ile Lys Gly Ile Ser Gly                 245 250 255 Asp Phe Ala Ala Lys Ser Glu Val Leu Asn Met Lys Phe Lys Asp Lys             260 265 270 Asn Asp Thr Ser Thr Phe Arg Gly Thr Leu Gly Ala Ser Ala Gln Leu         275 280 285 Leu Glu Asn Phe Glu Val His Leu Asp Ile Phe Gly Asp Leu Gly Asn     290 295 300 Asp Lys Gly Ile Gly Gly Gln Val Gly Ala Thr Tyr Arg Phe 305 310 315 <210> 3 <211> 294 <212> PRT <213> Lawsonia intracellularis <400> 3 Met Ser Leu Val Ile Asn Asn As Met Met Ala Ala Asn Ala Ala Arg   1 5 10 15 Asn Leu Asn Glu Ser Ser Ser Arg Ser Leu Ser Ser Ser Arg Ser Leu              20 25 30 Ser Ser Gly Leu Arg Val Gly Thr Ala Ala Asp Asp Ser Ala Gly Leu          35 40 45 Ala Ile Arg Glu Leu Met Arg Ala Asp Ile Lys Thr Phe Gln Gln Gly      50 55 60 Ala Arg Asn Ala Asn Asp Ala Ile Ser Leu Val Gln Val Ala Asp Gly  65 70 75 80 Ala Leu Gly Val Ile Asp Glu Lys Leu Ile Arg Met Lys Glu Leu Ala                  85 90 95 Glu Gln Ala Ala Thr Gly Thr Tyr Asn Ser Thr Gln Arg Leu Ile Ile             100 105 110 Glu Ser Glu Tyr Gln Ala Met Ala Ser Glu Ile Thr Arg Ile Ser Val         115 120 125 Ala Thr Glu Phe Asn Gly Ile Lys Leu Leu Asp Gly Ser Leu Ser Gly     130 135 140 Pro His Lys Gly Thr Asn Leu Gln Gln Thr Gly Ala Leu Arg Val His 145 150 155 160 Phe Gly Pro Gly Asn Ser Ser Ala Glu Asp Tyr Tyr Glu Ile Ser Ile                 165 170 175 His Ser Ala Thr Ala Ser Ala Leu Gly Leu Gly Asn Gly Thr Thr Gly             180 185 190 Pro Gly Ala Thr Ile Ser Thr Gln Ala Ala Ala Gln Ala Ala Leu Asp         195 200 205 Ala Ile Asn Ale Ile Val Ser Lys Asp Asn Ile Arg Ala Ser Leu     210 215 220 Gly Thr Leu Gln Asn Arg Leu Glu Ala Thr Ile Thr Asn Leu Asn Thr 225 230 235 240 Gln Ala Glu Asn Leu Glu Ala Ala Glu Ser Arg Ile Ser Asp Ile Asp                 245 250 255 Val Ser Thr Glu Met Thr Glu Phe Val Arg Asn Gln Ile Leu Thr Gln             260 265 270 Ser Gly Val Ala Met Leu Ser Gln Ala Asn Ser Leu Pro Lys Met Ala         275 280 285 Ser Gln Leu Ile Ser Gly     290 <210> 4 <211> 251 <212> PRT <213> Lawsonia intracellularis <400> 4 Met Ala Lys His Lys Val Arg Ala Asp Glu Leu Val Phe Leu Gln Gly   1 5 10 15 Leu Ala Glu Ser Arg Glu Gln Ala Lys Arg Leu Ile Met Ala Gly Lys              20 25 30 Val Thr Leu Thr Asn Asn Ser Thr Thr Ile Pro Leu Arg Leu Glu Lys          35 40 45 Pro Gly His Lys Tyr Pro Leu Glu Ser Ile Cys Ser Leu Ile Gly Val      50 55 60 Glu Arg Phe Val Ser Arg Gly Ala Tyr Lys Leu Leu Thr Ala Leu Asp  65 70 75 80 Phe Phe Lys Ile Asp Val Lys Ser Cys Ile Cys Leu Asp Ala Gly Ala                  85 90 95 Ser Thr Gly Gly Phe Thr Asp Cys Leu Leu Gln His Gly Ala Ser Lys             100 105 110 Val Tyr Ala Ile Asp Val Gly Lys Gly Gln Leu His Glu Lys Leu Tyr         115 120 125 Thr Asn Glu Gln Val Ile Asn Ile Glu Gly Val Asn Leu Arg Thr Ala     130 135 140 Ser Lys Asp Leu Ile Pro Glu Glu Val Asp Ile Leu Thr Ile Asp Val 145 150 155 160 Ser Phe Ile Ser Leu Thr Leu Ile Leu Pro Ser Cys Ile Arg Trp Leu                 165 170 175 Lys Ala Ser Gly Ile Ile Ile Ale Leu Ile Lys Pro Gln Phe Glu Leu             180 185 190 Tyr Pro Asp Lys Ile Lys Lys Gly Val Val Lys Glu Thr Ser Leu Gln         195 200 205 Tyr Glu Ala Val Glu Lys Ile Ile His Phe Cys Gln Ser Glu Leu Gly     210 215 220 Leu Ile Phe Ile Gly Val Val Pro Ser Val Ile Lys Gly Pro Lys Gly 225 230 235 240 Asn Gln Glu Tyr Leu Ile Tyr Leu Lys Lys Arg                 245 250 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gtcgacattt ttaatcctca agat 24 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ctcgagttag aatctataag tagca 25 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gtcgacattt ttaatcctca agat 24 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ctgcagttag aatctataag tagca 25 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ccgaattctc tttggtcatt aacaacaaca tga 33 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ccgtcgacgc cactaatgag ttggcttg 28 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gaattcgcca aacataaagt acgtgctgat 30 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 aagcttacgt tttttcaagt aaataagata ttcttg 36 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tcaagtggca tagatgtgga agaa 24 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tggctctgca ggattttcat g 21 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 acaggagaag ggacgccat 19 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gaagccctac agacgagctc a 21 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 accgcaatga agaccctgat 20 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tccctccgca ttgacaca 18

Claims (9)

delete delete delete delete delete delete A vaccine composition for the simultaneous prevention or treatment of porcine proliferative ileitis and salmonellosis comprising as an active ingredient a mixture of attenuated Salmonella mutants comprising all of the attenuated Salmonella variants below,
Each attenuated Salmonella mutant is a strain of Lawsonia intracellularis in which the lon , cpxR , rfaL and asd genes are deleted and consists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: Wherein the vaccine composition is an attenuated salmonella mutant comprising any one of the antigens selected from the group consisting of OptA, OptB, FliC, and Hly antigens derived from E. coli. 8. The vaccine composition according to claim 7, wherein the vaccine composition is orally or subcutaneously administered to a vaccine composition for preventing or treating swine proliferative ileitis and salmonellosis. delete

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