KR102107519B1 - Vaccine composition for prevention or treatment of brucellosis comprising InpB, Dps, AspC and Ndk protein derived from Brucella abortus as effective component - Google Patents

Abstract

The vaccine composition for preventing or treating brucellosis infection comprising InpB, Dps, AspC, and Ndk proteins derived from the brucellula avotus strain as an active ingredient, and the vaccine composition of the present invention can be widely used for prevention or treatment of brucellosis infection will be.

Description

Vaccine composition for prevention or treatment of brucellosis comprising InpB, Dps, AspC and Ndk protein derived from Brucella abortus as effective component}

The present invention is to prevent brucellosis infection including InpB (invasion protein B), Dps (DNA starvation / stationary phase protection protein), AspC (aspartate aminotransferase) and Ndk (nucleoside diphosphate kinase) protein derived from Brucellella avotus strain as an active ingredient or It relates to a therapeutic vaccine composition.

Brucella infectious disease is one of the representative common infectious diseases (human body; 4th legislative disease, livestock; 2nd legislative disease) and is known to cause serious public health and economic problems both domestically and globally. Recently, brucellella infections seen in Korea are showing an explosive increase in humans and livestock, and it is urgent to take measures for this as well as national concerns.

Brucella abortus (cow), B. melitensis (sheep, goat), B. canis (dog), B. ovis (sheep), B. suis (pig) are known to cause brucellosis. Infection in the human body can lead to serious illness, and in Korea, Brucella abortus is known to account for a high proportion. When brucella infection develops, the human body has a fatality rate of less than 10%, and persistent fever, headache, loss of appetite, weakness, progress to severe diseases such as endocarditis, encephalomyelitis, and arthritis. Symptoms seen in infected animals Is known to cause infertility due to abortion due to placenitis, endometritis, etc. in females, and orchitis, malformed sperm, etc. in males, without special external symptoms. Since brucela bacteria multiply in the cell and cause onset, in the case of human infection, antibiotic treatment for several weeks to several years is required, and improvement of treatment methods is urgently needed due to sequelae caused by taking large amounts of antibiotics. Even if it is judged to be cured, it often recurs within a few years, and continuous observation is required.

Since Brucella is an intracellular pathogen that relies primarily on cell-mediated immunity (CMI), the resistance of the host has been obtained, a live attenuated vaccine capable of stimulating potent cell-mediated immunity against Brucella infection It is mainly used. Although vaccination is the most economical means of infection control, currently available live attenuated vaccines such as Brucella Avotus S19 (smooth) or RB51 (rough) strains are not sufficient to eliminate Brucellosis infection in certain host species. This was confirmed. Moreover, live attenuated vaccines have several drawbacks, such as abortion and excretion of vaccine strains into milk. Therefore, other approaches are needed for the development of efficient, safe and effective vaccines, and immunoassays using recombinant proteins are attracting attention as promising candidates.

So far, L7 / L12 ribosome protein, Cu-Zn superoxide dismutase (SOD) protein, 22.9-kDa protein, lumazine synthase, outer membrane proteins Omp31, Omp16 or Omp19, and diphosphate nucleosides against pathogenic Brucella infection Numerous studies have been reported on the protective effects of various types of recombinant Brucella proteins, including phosphatase (nucleoside diphosphate kinase, NDPK). However, administration of a single antigen does not effectively induce an immune response in mice from infection of Brucella bacteria, so it is expected that combinations of different types of antigen proteins will show a higher protective effect than a single antigen.

On the other hand, Korean Registered Patent No. 1840360 is a vector developed to increase the secretion of BLS, Omp19, PrpA, and SOD antigens derived from Brucella Avotus, and contains attenuated Salmonella, which has an attenuated L-O-antigen of LPS as an active ingredient. A vaccine composition for preventing or treating brucellosis, comprising an O-antigen deletion non-pathogenic Salmonella strain of LPS expressing a major common antigen of Brucella, is disclosed, and Korean Patent No. 1775369 discloses an 'Ohr recombinant protein derived from a Brucellella avotus strain A vaccine composition for preventing or treating brucellosis infection comprising as an active ingredient 'is disclosed, but a vaccine for preventing or treating brucellosis infection comprising InpB, Dps, AspC, and Ndk proteins derived from the brucellella avotus strain of the present invention as an active ingredient There is no description of the composition.

The present invention has been derived by the above-mentioned needs, the present inventors in order to compensate for the drawbacks of subunit vaccines known to be less than live vaccines in terms of immunogenicity and infection defense effect, derived from the Brucella abortus strain A recombinant protein of high immunogenic proteins InpB, Dps, AspC, and Ndk was prepared, and the infection defense effect was confirmed by using a subunit vaccine containing all four recombinant proteins and a subunit vaccine containing each recombinant protein. As a result, when challenged with pathogenic Brucella bacteria in a group immunized with a subunit vaccine containing all four recombinant proteins, the number of pathogenic Brucella bacteria in the spleen was significantly reduced compared to a group immunized with a subunit vaccine containing only individual recombinant proteins. The present invention was completed by confirming that it was completed.

In order to solve the above problems, the present invention is derived from Brucella abortus ( Brucella abortus ) strain InpB (invasion protein B), Dps (DNA starvation / stationary phase protection protein), AspC (aspartate aminotransferase) and Ndk (nucleoside diphosphate kinase) ) It provides a subunit vaccine composition for the prevention or treatment of brucellosis infection containing protein as an active ingredient.

In addition, the present invention provides a method for preventing or treating brucellella infection, comprising administering the subunit vaccine composition to a livestock that is expected to develop brucellella infection or has developed brucellosis infection.

In addition, the present invention provides a feed composition for the prevention or improvement of brucellella infections comprising InpB, Dps, AspC and Ndk proteins derived from the brucellella avotus strain as an active ingredient.

The vaccine of the present invention is a subunit vaccine having better stability than a live vaccine, and the subunit vaccine of the present invention contains four high antigenic proteins derived from Brucellar abotus, and thus has excellent immunogenicity and protection against infection, thereby preventing brucellosis. Or it could be useful for treatment.

1 is a result of confirming the immunoreactivity of the recombinant protein derived from Brucella abotus, (A) is the result of staining the gel with Brilliant Blue after co-developing the purified protein by SDS-PAGE, and (B) (C) is a result of performing Western blot with mouse serum positive or negative, respectively. Lane 1; MBP, Lane 2; Marker, lane 3; rDps, lane 4; rInpB, lane 5; rYaeC, Lane 6; rAspC.
2 is a result of confirming the humoral immune response induced after immunizing mice with each recombinant protein.
3 is a result of confirming the humoral immune response induced after immunizing mice with CSV (a combination subunit vaccine of InpB, Dps, AspC and Ndk).
4 is a result of analyzing the CD4 ⁺ and CD8 ⁺ T cell population in the blood of mice immunized with CSV.

In order to achieve the object of the present invention, the present invention is derived from Brucella abortus ( Brucella abortus ) strain InpB (invasion protein B), Dps (DNA starvation / stationary phase protection protein), AspC (aspartate aminotransferase) and Ndk (nucleoside) It provides a subunit vaccine composition for the prevention or treatment of brucellosis infection containing diphosphate kinase) protein as an active ingredient.

In the present invention, the term "subunit vaccine" refers to a vaccine comprising an infectious defense antigen isolated from a microorganism, and usually refers to a protein subunit vaccine that uses only some proteins of a microorganism by protein purification or genetic recombination technology. do.

The range of InpB protein, Dps protein, AspC protein and Ndk protein according to the present invention is a protein having an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and a functional equivalent of the protein. Includes. As a result of addition, substitution, or deletion of an amino acid, a "functional equivalent" means at least 70%, preferably 80% or more of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, More preferably, it has a sequence homology of 90% or more, and even more preferably 95% or more, and has substantially the same physiological activity as the protein represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. Refers to the indicated protein

The term "prevention" in the present invention means any action that suppresses or delays the onset of brucellosis infection by administration of the vaccine composition of the present invention.

The term "treatment" in the present invention means any action that improves or benefits the symptoms of brucellosis infection already caused by administration of the vaccine composition of the present invention.

The vaccine composition of the present invention further comprises a pharmaceutically acceptable carrier or diluent. Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. Such carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous solutions carriers include water, alcohols / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous carriers include electrolyte supplements such as those based on Ringer's dextrose, liquid and nutrient supplements, and the like. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases and the like may further be present. Preferred preservatives include formalin, thimerosal, neomycin, polymyxin B and amphotericin B.

In addition, the vaccine composition of the present invention may further include an adjuvant (adjuvant, adjuvant, adjuvant). The adjuvant refers to a compound or mixture that enhances the immune response and / or promotes the rate of absorption after inoculation, and includes any absorption-promoting agent. Acceptable adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, fluron polyols, polyanions, peptides, oil or hydrocarbon emulsions. , Keyhole limpet hemocyanin, dinitrophenol, and the like.

The vaccine composition of the present invention may be administered through any one administration route selected from oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or nasal passages, and may be administered by bolus or slowly injected. It is preferably administered by injection.

The vaccine composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention is an amount sufficient to exhibit a vaccine effect, and means an amount that does not cause side effects or serious or excessive immune responses, and the effective dose level is a disorder to be treated. , The severity of the disorder, the activity of the specific compound, the route of administration, the rate of removal of the recombinant proteins (rInpB, rDps, rAspC and rNdk), the duration of treatment, the drug used in combination with or concurrently with the recombinant protein, age, weight, sex of the subject, It can depend on a variety of factors, including eating habits, general health conditions and factors known in the medical arts. In determining the "therapeutically effective amount", various general considerations that are considered are known to those skilled in the art.

In a method according to an embodiment of the present invention, a method capable of producing InpB, Dps, AspC and Ndk proteins, SEQ ID NO: 5 (InpB), SEQ ID NO: 6 (Dps), SEQ ID NO: 7 (AspC) or SEQ ID NO: It is preferable that the expression vector containing the gene of 8 (Ndk) is transformed into a host cell and then purified by expressing a large amount of InpB , Dps , AspC or Ndk gene, but is not limited thereto.

The gene of the present invention includes both genomic DNA and cDNA encoding InpB, Dps, AspC or Ndk proteins. Preferably, the gene of the present invention may include a nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. In addition, homologs of the base sequences are included within the scope of the present invention. Specifically, the gene and the base sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, respectively, 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably May include a base sequence having at least 95% sequence homology. “% Of sequence homology” to a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It does not include) may be added or deleted (ie, gap).

The term “vector” is used to refer to DNA fragment (s), nucleic acid molecules that are delivered into a cell. Vectors replicate DNA and can be independently reproduced in host cells. The term "expression vector" is often used interchangeably with "recombinant vector." The term "recombinant vector" refers to a recombinant DNA molecule comprising the desired coding sequence and the appropriate nucleic acid sequence necessary to express the linking coding sequence operably in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

The term "recombinant" in the present invention refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide or a protein encoded by a heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the natural form of the cells, either in sense or antisense form. Recombinant cells can also express genes found in natural cells, but these genes have been modified and reintroduced into cells by artificial means.

The present invention also provides a method for preventing or treating brucellella infection, comprising administering the subunit vaccine composition to a livestock that is expected to develop brucellella infection or has developed brucellella infection.

The term "livestock" of the present invention is an animal that can develop brucellosis infection, and preferably may be livestock such as cows, pigs, sheep, goats, dogs, but is not limited thereto.

When the subunit vaccine composition of the present invention is administered to the livestock, the immunity of an individual against a strain causing brucellosis infection may be enhanced, thereby preventing or treating brucellosis infection.

In addition, the present invention is also an active ingredient of InpB (invasion protein B), Dps (DNA starvation / stationary phase protection protein), AspC (aspartate aminotransferase) and Ndk (nucleoside diphosphate kinase) protein derived from Brucella abortus strain It provides a feed composition for the prevention or improvement of brucellosis infection containing.

The InpB protein, Dps protein, AspC protein and Ndk protein according to the present invention may be composed of amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the range is as described above.

The present invention may include general feed ingredients known in the art. For example, the feed composition of the present invention may include grain flour, sugar, vitamins, amino acids, proteins, lipids, minerals, and the like.

Grain and grain by-products may be used in the feed composition of the present invention, such as rapeseed, cottonseed, soybean, wheat bran, rice bran, defatted river, malt, corn bran, malt, soybean skin, potato starch, sweet potato starch, corn starch, Coffee foil, jamsa, jam, seaweed, tapioca, soybean meal, cottonseed meal, gourd, chaejong, flax, zucchini, corn gluten, wheat gluten, peanut melon, palm melon, sunflower seed melon, drunk melon, corn germ , One or two or more selected from pepper seed meal, jangyu-bak, and beer-bak can be mixed.

The feed composition of the present invention may include sugar and complex carbohydrates such as water-soluble and insoluble monosaccharides, disaccharides and polysaccharides. More specifically, as sugars that can be used, glucose, mannose, fructose, white sugar, maltose, cellobiose, lactose, trehalose, melibiose, raffinose, escreen, salicin, amidaline, mannitol, sorbitol, Sorbose, mentiose, and the like, and can be used in combination with oligosaccharides as well as molasses and sucrose.

Arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine, alanine, aspartic acid, sodium glutamate, glycine, proline, serine , Cysteine, and their homologues, and salts thereof.

In addition, thiamin (vitamin B1), HCl, riboflavin (vitamin B2), pyridoxine (vitamin B6), HCl, niacin (vitamin B3), niacinamide (water soluble vitamin and vitamin B) that can be optionally added to the feed composition of the present invention Complex), vitamin B groups such as inositol (vitamin B8), choline chloride (vitamin B4), calcium pantothenate (vitamin B5), biotin (vitamin B7), folic acid (vitamin B9) and vitamins A, C, K, D and E And so on. Among them, vitamin A, vitamin B group, and vitamin E can also be used as antioxidants.

Fatty acids that may be included in the feed composition of the present invention include, for example, vegetable oils such as soybean oil, rapeseed oil, corn oil, safflower oil, sunflower oil, rice oil, beefsteak vegetable oil, evening primrose oil, glass fat oil, linseed oil, And fish oil such as bonito, mackerel, sardine, and triglycerides derived from various microorganisms, by hydrolysis treatment, and calcium salts and magnesium salts of fatty acids may also be included as metal salts of fatty acids obtained above. In the feed composition of the present invention, a small amount of a known bioceramic material such as plagioclase, bentonite, and mackban stone can be added to double the antibacterial function.

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

Materials and methods

1. Bacterial strains and growth conditions

Smooth type, pathogenic Brucella Abbots abortus ) 544 The biovar 1 strain was used by pre-sale from the Animal, Plant and Fisheries Quarantine and Inspection Agency, and the E. coli DH5α strain was purchased from Invitrogen (USA). The cultivation of Brucella abotus bacteria was cultured to a stationary phase at 37 ° C using Brucella culture medium (BD Biosciences, USA), and the E. coli strain used LB liquid or solid medium containing 100 μg / ml of ampicillin (Sigma, USA) And cultured at 37 ° C.

2. Preparation of plasmid and purification of recombinant protein

Cloning and protein purification of the InpB , Dps , AspC and Ndk genes of Brucella Abbotus was performed according to the method of the previous study (Hop HT. Et al., FEMS Microbiol Lett. 2015, 362 (4)). Briefly, a gene-specific primer set was used to clone the full length of each Brucella Abbotus gene into a pMAL vector (Table 1). E. coli cells transformed with a vector containing each gene were cultivated to produce a recombinant protein, and the amount of IPTG (Isopropyl β-D-1-thiogalactopyranoside) for inducing protein expression during culture is presented in Table 1 below.

IPTG usage for inducing primer set and protein expression gene Forward (5 '→ 3') (SEQ ID NO) Reverse (5 '→ 3') (SEQ ID NO) IPTG (mM) InpB AGC GGATCC ATGAAAAATTATCG3 (9) ATC CTGCAG TTACTTGGTCAATGC (10) 0.4 Dps AGC GGATCC ATGCCGAAGAAGTC (11) ATC CTGCAG TTAATTGCTTTCCTG (12) 0.1 AspC AGC GGATCC ATGGCATTTCTCGCC (13) ATC CTGCAG TCAGCGCAGGCTGGC (14) 2.0 Ndk CGC GGATCC ATGGCAATTGAACG (15) GGC CTGCAG TCAGCCAACGATTTC (16) 0.2 YaeC AGC GGATCC ATGTCGTCAGTCCTT (17) ATC CTGCAG TTAATTCCAGGCCGG (18) 1.0

(Underlined: BamH I and Pst I restriction enzyme sites)

Purification of the recombinant protein was performed by centrifuging the cultured cells at a rate of 3,000x g for 10 minutes to collect cell pellets, resuspending with 20 ml of column buffer, and freeze-thaw at -70 ° C / 4 ° C. After repeating the procedure three times, the cells were placed in an ice bucket and ultrasonically crushed under conditions of 10,000 Hz. Thereafter, the supernatant was recovered by centrifugation for 20 minutes at a rate of 5,000x g , and the recombinant protein was purified by a maltose resin column (Bio-Rad) using a column buffer containing 10 mM maltose as eluent.

3. SDS-PAGE and Western blot

Cell eluates from which protein expression was induced and purified recombinant proteins were analyzed by SDS-PAGE and Western blot. Brucella Abbotus-negative or positive mouse serum was used as the primary antibody.

4. Mouse immunization and bacterial challenge

To test the protective effect of each individual antigen, 35 female BALB / c mice (Japan SLC, Japan), 6 weeks of age, were divided into 7 groups. Each mouse was mixed with 10 μl of protein (MBP, InpB, Dps, AspC or Ndk) or PBS in 100 μl of Incomplete Freund's adjuvant (Sigma), at week 0, 2, 4 and 6 Immunization was performed by intraperitoneal administration.

To confirm the protective effect of the mixed subunit vaccine (InpB, Dps, AspC and Ndk combined subunit vaccine, CSV), 20 mice were randomly divided into 4 groups, and then 100 μl of Freund's incomplete adjuvant. Immunization was induced by intraperitoneal administration at week 0, 2, 4 and 6 by mixing 20 μg of mixed protein (InpB, Dps, AspC and Ndk mixed in a ratio of 1: 1: 1: 1) or PBS. . As a positive control, 5 × 10 ⁶ CFU RB51 (/ 100 μl PBS) was used by intraperitoneal administration on day 0. Both individual antigen and mixed subunit vaccine immunization groups were collected from the tail vein of each mouse at week 8 to obtain a serum sample, and each mouse was administered intraperitoneally with 5 × 10 ⁵ CFU Brucella Avotus (/ 100 μl PBS). Was challenged.

5. CD4 in peripheral blood ⁺  And CD8 ⁺  Quantification of T cells

A population of cells in peripheral blood drawn from mice was performed according to the previous method (Yang Z. et al., Vaccine 2007, 25: 161-9). To 100 μl of blood collected from each mouse, 75 μl of FITC-linked rat anti-mouse CD4 and PE-linked rat anti-mouse CD8 monoclonal antibody (SantaCruz Biotechnology, USA) were added, and reacted for 30 minutes at cancer conditions and room temperature. . Thereafter, 1 ml of red blood cell lysis buffer was added and reacted at 37 ° C for 30 minutes. The reaction was terminated by adding 2 ml of PBS, centrifuged at 4 ° C., 380 x g, for 5 minutes , the supernatant was discarded, the cell pellet was washed twice with PBS, and resuspended with 0.5 ml of PBS, followed by FACS Calibur flow cytometry. Analysis was done with an analyzer (BD Biosciences).

6. ELISA and cytokine quantification

The titers of IgG1 and IgG2a in serum specific to the recombinant protein were performed through ELISA analysis with reference to the method of Luo et al. (Infect Immun. 20016, 74: 2734-41). The cutoff value was calculated by adding the mean and standard deviation of the OD values based on non-immunized mouse serum diluted 1: 100. Levels of IL-10, IL-12p70 and INF-γ in serum were analyzed using a cytometric bead array (BD CBA Mouse Inflammation Kit).

7. Infection protection experiment

The infection protection study was performed by partially modifying the method of Luo et al. After 4 weeks of bacterial infection, the spleen was harvested at the expense of mice, weighed, and homogenized in PBS conditions. The homogenized solution was serially diluted 10-fold using PBS, and then incubated for 3 days at 37 ° C. on a Brucella solid medium. The log ₁₀ CFU was calculated for each sample, and the protective effect was calculated by subtracting the log ₁₀ CFU value of each experimental group from the log ₁₀ CFU of the PBS group. All animal experiments were conducted in accordance with the guidelines of the Animal Ethics Committee at Gyeongsang National University (approval number GNU-170331-M0017).

All the results of the experiment were expressed as the mean ± standard deviation, and were tested by one-way ANOVA.

Example 1. Purification and immunogenicity analysis of recombinant protein

As a result of confirming the size and immunogenicity of each protein by deploying each purified recombinant protein on a 10% SDS-PAGE gel, the recombinant proteins rDps, rInpB, rYaeC and rAspC showed molecular weights of 61.92, 63, 74 and 87.9 kDa, respectively. (Fig. 1A), and at the same time, showed a strong reactivity to Brucella abotus-positive serum, but it was confirmed that the immune response did not occur in Brucella abotus-negative serum (Figs. 1B and 1C).

Example 2. Analysis of immune response following administration of single antigen

Previous studies have confirmed that the immune response peaks after three independent immunizations with recombinant proteins. In the present invention, after three independent antigen protein (subunit vaccine) inoculations, serum samples of mice were obtained to analyze the immune response. As a result, it was found that serum samples immunized with rDps, rInpB, rYaeC, and rAspC induced higher humoral immune responses than serum samples immunized with PBS or maltose binding protein (MBP) (FIG. 2). In addition, it was confirmed that the titer of IgG2a was higher than that of IgG1, and thus the rDps, rInpB and rAspC proteins of the present invention could be inferred that the Th1 response induces a predominant immune response. However, the rYaeC protein was observed to have a higher titer of IgG1 than IgG2a, so the rYaeC protein could be inferred that the Th2 response induces a predominant immune response.

In order to confirm the cell-mediated immune response, as a result of analyzing the levels of cytokines in the serum, production of IL-10, IL-12p70 and INF-γ is induced in mice immunized with MBP as shown in Table 2 below. On the other hand, production of IL-10 and INF-γ was induced in mice immunized with rDps, rInpB and rAspC, and IL-12p70 was confirmed only in serum of mice immunized with rDps and rAspC. The rYaeC protein was found to have weaker induction of cytokine production than other proteins.

Analysis of cytokine levels in mouse serum immunized with a single subunit vaccine Vaccine antigen Cytokine concentration (pg / ml) INF-γ IL-10 IL-12p70 PBS not detectable not detectable 0.16 ± 0.03 MBP 0.28 ± 0.08 not detectable 0.21 ± 0.06 rDps 6.47 ± 0.44 1.42 ± 0.05 3.31 ± 0.29 rInpB 5.13 ± 0.73 2.42 ± 0.04 0.26 ± 0.12 rYaeC 1.77 ± 0.41 0.92 ± 0.50 0.10 ± 0.14 rAspC 8.62 ± 0.30 2.66 ± 0.22 5.47 ± 0.64

Example 3. Analysis of protective effect against brucellella challenge in single subunit vaccinated mice

In order to confirm the vaccine effect of the single recombinant protein (antigen), 2 weeks after the immunization reaction, Brucella avotus bacteria were challenged, and 4 weeks after bacterial infection, mice were sacrificed to determine the number of challenged bacteria in the spleen. As a result, as shown in Table 3, in the rDps, rInpB, and rAspC subunit vaccination group, it was found that the number of challenge-infected bacteria was reduced at a significance level compared to the PBS treatment group, and the log protection effect compared to the PBS treatment group, respectively. It was found that it was 1.35, 1.13 and 1.48 times higher. The group vaccinated with the MBP and rYaeC proteins was found to have no protective effect against the challenge-infected strains compared to the PBS-treated group, similar to the results of the immune response analysis of Example 1.

Challenge infection results of mice immunized with a single subunit vaccine Vaccine antigen Number of brucellella in the spleen
(log ₁₀ , mean ± standard deviation) log protection effect valence PBS 5.79 ± 0.36 MBP 5.65 ± 0.17 0.14 p> 0.05 rDps 4.44 ± 0.12a 1.35 p <0.01 rInpB 4.66 ± 0.10a 1.13 p <0.01 rYaeC 5.34 ± 0.19 0.45 p> 0.05 rAspC 4.31 ± 0.25a 1.48 p <0.01

Example 4. Analysis of humoral immune response in immunized mice with mixed subunit vaccine

Through the above results, it was confirmed that the InpB, Dps, and AspC proteins are high-immunity antigens, and Ndk proteins identified through our previous studies (Hop HT. Et al., FEMS Microbiol Lett. 2015, 362 (4)) In combination, a vaccine against brucellella infection was prepared.

First, the immune response of mice immunized with a mixed subunit vaccine (a combination subunit vaccine of InpB, Dps, AspC and Ndk, hereinafter CSV) was analyzed. The immune response of CSV was analyzed using serum after 1 vaccine administration and serum after 3 vaccine administrations. As a result, the group immunized with CSV showed an active IgG response after one immunization compared to the group immunized with MBP, and it was found that the titer of IgG increased as the number of vaccination increased (FIG. 3). Although the group immunized with live vaccine (RB51) was found to have a higher humoral response than the group immunized with CSV, the live vaccine group had a ratio of IgG2a / IgG1 less than 1, whereas the group immunized with CSV As the ratio of IgG2a / IgG1 was confirmed to be high at 1.2, it was inferred that the Th1 response was the predominant immune response. Humoral immunity was not induced in the PBS-injected group (without results).

In addition, as a result of analyzing the level of cytokines in each group, as shown in Table 4 below, the group injected with PBS did not induce the generation of cytokines, but the group immunized with RB51 or CSV was also IL-10 after one vaccination. , IL-12p70 and INF-γ production was found to be induced. In addition, it was found that the group immunized with MBP did not induce cytokine production after one vaccination. In particular, the group immunized with CSV showed that the levels of IL-10, IL-12p70, and INF-γ gradually increased until 3 inoculations, and in particular, the level of INF-γ was significantly increased, while immunized with RB51 The group found that the level of cytokines analyzed at week 2 was slightly reduced at week 8.

Analysis of cytokine levels in mouse serum immunized with a mixed subunit vaccine Cytokine concentration (pg / ml) INF-γ IL-10 IL-12p70 weeks 2 8 2 8 2 8 PBS ND ND ND ND ND ND MBP ND 0.14 ± 0.16 ND ND ND 0.15 ± 0.13 RB51 14.2 ± 1.03 6.72 ± 0.89 4.18 ± 0.29 3.24 ± 0.36 2.51 ± 0.34 1.67 ± 0.18 CSV 2.17 ± 0.42 17.32 ± 1.27 0.94 ± 0.11 3.08 ± 1.04 0.84 ± 0.20 4.63 ± 1.21

(ND: not detectable)

Example 5. CD4 in peripheral blood with mixed subunit vaccine ⁺  And CD8 ⁺  T cell subset analysis

CD4 ⁺ and CD8 ⁺ T cells are known to play an important role in cellular immunity, and are known to make important contributions to Brucella resistance. In the present invention, a subset of T cells in peripheral blood by vaccination was analyzed using a flow cytometer.

As a result, PBS and MBP treatments were found to have no change in the population level of CD4 ⁺ and CD8 ⁺ T cells, but it was found that the group immunized with RB51 or CSV induced CD4 ⁺ and CD8 ⁺ T cell differentiation. (Figure 4). In addition, in the group immunized with CSV, high-level CD4 ⁺ and CD8 ⁺ T cell populations were confirmed up to Weeks 2 and 8, whereas the group immunized with RB51 was found to have CD4 ⁺ and CD8 ⁺ T cell populations highly identified in Week 2. At week 8, it was observed that it was reduced to a level similar to the PBS treatment.

Example 6. Analysis of protective effect against brucellella challenge in mixed subunit vaccinated mice

As a result of analyzing the vaccine effect of CSV in the same manner as in Example 3, it was found that the CSV of the present invention shows excellent protective effect against Brucella bacteria (Table 5).

Challenge infection results of mice immunized with the mixed subunit vaccine Vaccine antigen Number of brucellella in the spleen
(log ₁₀ , mean ± standard deviation) log protection effect valence PBS 5.23 ± 0.48 MBP 5.17 ± 0.26 0.06 p> 0.05 RB51 2.92 ± 0.15a 2.31 p <0.01 CSV 3.04 ± 0.13a 2.19 p <0.01

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Vaccine composition for prevention or treatment of brucellosis          comprising InpB, Dps, AspC and Ndk protein derived from Brucella          abortus as effective component <130> PN18144 <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 173 <212> PRT <213> Brucella abortus <400> 1 Met Lys Asn Tyr Arg Ala Ile Gly Leu Ala Phe Thr Phe Thr Ala Leu   1 5 10 15 Ser Ser Leu Ser Ala Phe Ala Ala Ser Leu Pro Gly Gly Ala Ser Thr              20 25 30 Leu Gln Glu Thr Tyr Gln Asp Trp Thr Val Ser Cys Gln Ser Gln Lys          35 40 45 Asp Thr Thr Ala Cys Val Met Arg Gln Glu Gln Ser Ser Ala Gln Thr      50 55 60 Gly Gln Arg Val Leu Thr Ala Glu Leu Arg Asn Val Ala Gly Gly Lys  65 70 75 80 Val Asp Gly Val Leu Leu Met Pro Phe Gly Leu Asp Leu Ala Lys Gly                  85 90 95 Ala Ser Leu Lys Ile Asp Asp Thr Ala Gly Pro Asn Leu Thr Phe Ser             100 105 110 Thr Cys Leu Pro Gln Gly Cys Leu Ala Pro Val Ser Phe Asp Ala Lys         115 120 125 Gln Val Ala Ala Leu Lys Ser Gly Thr Asn Ile Asn Val Thr Thr Thr     130 135 140 Ala Leu Ser Pro Ser Gln Pro Val Ala Phe Lys Ile Ser Leu Lys Gly 145 150 155 160 Phe Gly Ala Ala Leu Asp Arg Ile Gln Ala Leu Thr Lys                 165 170 <210> 2 <211> 165 <212> PRT <213> Brucella abortus <400> 2 Met Pro Lys Lys Ser Met His Ala Thr Arg Asn Asp Leu Pro Ser Asn   1 5 10 15 Thr Lys Thr Thr Met Ile Ala Leu Leu Asn Glu Asn Leu Ala Ala Thr              20 25 30 Ile Asp Leu Ala Leu Ile Thr Lys Gln Ala His Trp Asn Leu Lys Gly          35 40 45 Pro Gln Phe Ile Ala Val His Glu Met Leu Asp Gly Phe Arg Ala Glu      50 55 60 Leu Asp Asp His Val Asp Thr Ile Ala Glu Arg Ala Val Gln Ile Gly  65 70 75 80 Gly Thr Ala Tyr Gly Thr Thr Gln Val Val Val Lys Glu Ser Arg Leu                  85 90 95 Lys Pro Tyr Pro Thr Asp Ile Tyr Ala Val His Asp His Leu Val Ala             100 105 110 Leu Ile Glu Arg Tyr Gly Asp Val Ala Asn Leu Val Arg Lys Ser Ile         115 120 125 Lys Asp Ala Asp Asp Ala Gly Asp Asp Asp Thr Ala Asp Ile Phe Thr     130 135 140 Ala Ala Ser Arg Ser Leu Asp Lys Ala Leu Trp Phe Leu Glu Ala His 145 150 155 160 Val Gln Glu Ser Asn                 165 <210> 3 <211> 400 <212> PRT <213> Brucella abortus <400> 3 Met Ala Phe Leu Ala Asp Ala Leu Ser Arg Val Lys Pro Ser Ala Thr   1 5 10 15 Ile Ala Val Ser Gln Lys Ala Arg Glu Leu Lys Ala Lys Gly Lys Asp              20 25 30 Val Ile Val Leu Gly Ala Gly Glu Pro Asp Phe Asp Thr Pro Glu Asn          35 40 45 Ile Lys Gln Ala Ala Ile Ala Ala Ile Asn Arg Gly Glu Thr Lys Tyr      50 55 60 Thr Pro Val Ser Gly Ile Pro Gln Leu Arg Gln Ala Ile Val Ser Lys  65 70 75 80 Phe Lys Arg Glu Asn Gly Leu Asp Tyr Lys Pro Glu Gln Thr Ile Val                  85 90 95 Gly Thr Gly Gly Lys Gln Ile Leu Phe Asn Ala Phe Met Ala Thr Leu             100 105 110 Asn Pro Gly Asp Glu Val Ile Ile Pro Ala Pro Tyr Trp Val Ser Tyr         115 120 125 Pro Glu Met Val Ala Ile Asn Gly Gly Thr Pro Val Phe Val Asp Thr     130 135 140 Lys Ile Glu Asp Asn Phe Lys Leu Thr Ala Ala Asp Leu Glu Lys Ala 145 150 155 160 Ile Thr Pro Lys Thr Lys Trp Leu Ile Phe Asn Ser Pro Ser Asn Pro                 165 170 175 Thr Gly Ala Ala Tyr Thr Gln Ala Glu Leu Lys Ser Leu Thr Asp Val             180 185 190 Leu Val Arg His Pro His Val Trp Ile Leu Thr Asp Asp Met Tyr Glu         195 200 205 His Leu Val Tyr Gly Asp Phe Val Phe Thr Thr Pro Ala Gln Val Glu     210 215 220 Pro Ser Leu Tyr Asp Arg Thr Leu Thr Met Asn Gly Val Ser Lys Ala 225 230 235 240 Tyr Ala Met Thr Gly Trp Arg Ile Gly Tyr Ala Ala Gly Pro Ile Glu                 245 250 255 Leu Ile Lys Ala Met Asp Met Ile Gln Gly Gln Gln Thr Ser Gly Ala             260 265 270 Cys Ser Ile Ala Gln Trp Ala Ala Val Glu Ala Leu Asn Gly Thr Gln         275 280 285 Asp Phe Ile Pro Ala Asn Lys Lys Ile Phe Gln Ala Arg Arg Asp Leu     290 295 300 Val Val Ser Met Leu Asn Gln Ala Thr Gly Leu Gln Cys Pro Thr Pro 305 310 315 320 Glu Gly Ala Phe Tyr Val Tyr Pro Ser Cys Ala Gly Leu Ile Gly Lys                 325 330 335 Lys Thr Glu Ala Gly Lys Val Ile Glu Thr Asp Lys Asp Phe Val Thr             340 345 350 Glu Leu Leu Glu Thr Glu Gly Val Ala Val Val His Gly Ser Ala Phe         355 360 365 Gly Leu Gly Pro Asn Phe Arg Ile Ser Tyr Ala Thr Ser Asp Glu Leu     370 375 380 Leu Glu Lys Ala Cys Ile Arg Ile Gln Arg Phe Cys Ala Ser Leu Arg 385 390 395 400 <210> 4 <211> 140 <212> PRT <213> Brucella abortus <400> 4 Met Ala Ile Glu Arg Thr Phe Ser Met Ile Lys Pro Asp Ala Thr Arg   1 5 10 15 Arg Asn Leu Thr Gly Ala Ile Ile Ala Lys Leu Glu Glu Ala Gly Leu              20 25 30 Arg Val Val Ala Ser Lys Arg Val Trp Met Ser Arg Arg Glu Ala Glu          35 40 45 Gly Phe Tyr Ala Val His Lys Asp Arg Pro Phe Phe Gly Glu Leu Val      50 55 60 Glu Phe Met Ser Ser Gly Pro Thr Val Val Gln Val Leu Glu Gly Glu  65 70 75 80 Asn Ala Ile Ala Lys Asn Arg Glu Val Met Gly Ala Thr Asn Pro Ala                  85 90 95 Asn Ala Asp Glu Gly Thr Ile Arg Lys Thr Phe Ala Leu Ser Ile Gly             100 105 110 Glu Asn Ser Val His Gly Ser Asp Ala Pro Glu Thr Ala Ala Glu Glu         115 120 125 Ile Ala Tyr Trp Phe Ser Gly Thr Glu Ile Val Gly     130 135 140 <210> 5 <211> 522 <212> DNA <213> Brucella abortus <400> 5 atgaaaaatt atcgtgcaat cggtcttgca ttcacgttca ctgccctttc cagtctttcg 60 gcctttgcag cctccctgcc cggcggagca agcaccttgc aggaaaccta tcaggactgg 120 accgtgtctt gccagtcgca gaaggataca acagcctgcg tgatgcgtca ggagcaaagc 180 agcgcccaga ccggccagcg cgttctgact gccgagctgc gcaacgtcgc cggcggcaaa 240 gttgacggtg tgttgctgat gccgttcggt cttgatcttg ccaagggcgc ctcgctcaag 300 attgatgaca ccgctgggcc aaacctcacc ttctccacct gcctgccgca gggctgcctc 360 gcgccagtga gcttcgatgc caagcaggtt gctgcgctga aatccggcac caacatcaat 420 gtcaccacga cggcgctcag cccgagccag ccggttgcct tcaagatttc cctgaaaggc 480 ttcggtgccg cgctcgaccg cattcaggca ttgaccaagt aa 522 <210> 6 <211> 498 <212> DNA <213> Brucella abortus <400> 6 atgccgaaga agtcgatgca tgcaacccgc aacgatcttc cctccaatac caagacgacg 60 atgatcgcgc tgctcaacga gaatcttgcc gcaacaatcg atcttgccct catcaccaag 120 caggcgcact ggaacctcaa gggaccgcaa ttcatcgccg tgcatgaaat gctcgatggt 180 ttccgcgcag aactcgacga ccatgtggac acgattgccg aacgtgccgt gcagatcggc 240 ggaacggcct atggtacaac ccaggtcgtg gtgaaggaat ccagactgaa gccttacccg 300 accgacattt atgccgtcca cgaccatctg gtggcactga tcgaacgcta tggtgatgtt 360 gccaatctgg tgcgcaaatc gatcaaggat gcagacgacg cgggtgacga cgacacggca 420 gatattttca ccgccgcatc gcgcagcctc gacaaggcac tctggttcct cgaagcgcat 480 gtgcaggaaa gcaattaa 498 <210> 7 <211> 1203 <212> DNA <213> Brucella abortus <400> 7 atggcatttc tcgccgacgc cctttcccgt gtaaagccat ctgcgaccat cgctgtcagc 60 cagaaagcac gtgaactgaa agcaaaaggc aaggacgtga tcgtcctggg cgcaggtgag 120 ccggatttcg acacgcctga aaatatcaag caggcggcca ttgccgcgat caatcgcggt 180 gaaacaaagt acacgcccgt atccggcatc ccgcaactgc gccaggccat cgtctccaag 240 ttcaagcgtg aaaacggcct cgactacaag ccggaacaga ccatcgtcgg caccggcggc 300 aagcagattt tgttcaacgc cttcatggcc acgctgaacc ccggcgacga agtcatcatt 360 cccgccccct attgggtcag ctacccggaa atggtggcca tcaacggcgg caccccggtt 420 ttcgtcgaca cgaagattga agacaatttc aagctgaccg ctgccgatct cgaaaaggcg 480 atcacaccga agaccaagtg gctcatcttc aactcgccct ccaacccgac cggcgcggcc 540 tatacgcaag ccgaactgaa gtcgctgacc gacgttctcg tgcgccatcc gcatgtctgg 600 atcttgaccg acgacatgta tgagcatctg gtctatggcg atttcgtctt cacgacgcct 660 gctcaggtgg aaccctcgct ctatgaccgt acgctcacca tgaacggcgt ttccaaggcc 720 tatgccatga ccggctggcg catcggctat gccgccggtc caatcgagct catcaaggcc 780 atggacatga ttcaggggca gcagacctcc ggtgcctgct cgattgctca gtgggcagcg 840 gtcgaagcac tgaacggcac gcaggatttc atccccgcga acaagaaaat cttccaggcc 900 cgccgcgatc tggtcgtttc catgctcaac caggcaaccg gcctccagtg cccgacgccg 960 gaaggcgcgt tctatgtcta tccgtcctgc gcagggctga tcggcaagaa gaccgaagcg 1020 ggcaaagtca tcgaaaccga caaagatttt gtgacggaac ttctggaaac cgaaggcgtg 1080 gcggttgttc atggttcggc attcggcctc ggcccgaact tccgcatttc ctatgcaacc 1140 tcggatgagc tgctggaaaa agcctgcata cgcatccagc gtttctgcgc cagcctgcgc 1200 tga 1203 <210> 8 <211> 423 <212> DNA <213> Brucella abortus <400> 8 atggcaattg aacgtacttt ctccatgatc aagcccgatg cgacccgccg caacctgact 60 ggcgcgatca tcgccaagct tgaagaagcg ggcctgcgcg ttgtcgcatc gaagcgcgtc 120 tggatgagcc gccgtgaagc tgaaggcttc tacgccgttc acaaggaccg tccgttcttt 180 ggcgaactgg ttgaattcat gtcctccggc ccgacggtcg ttcaggttct cgaaggcgaa 240 aacgcaattg ccaagaaccg tgaagtcatg ggcgccacca acccggccaa tgccgacgaa 300 ggcaccatcc gcaagacctt cgccctgtcc atcggtgaaa attcggttca cggttcggat 360 gctcccgaaa ccgctgccga agaaatcgcc tactggtttt cgggcaccga aatcgttggc 420 tga 423 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 agcggatcca tgaaaaatta tcg 23 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 atcctgcagt tacttggtca atgc 24 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 agcggatcca tgccgaagaa gtc 23 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 atcctgcagt taattgcttt cctg 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 agcggatcca tggcatttct cgcc 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 atcctgcagt cagcgcaggc tggc 24 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cgcggatcca tggcaattga acg 23 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ggcctgcagt cagccaacga tttc 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 agcggatcca tgtcgtcagt cctt 24 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 atcctgcagt taattccagg ccgg 24

Claims (6)

InpB (invasion protein B) consisting of the amino acid sequence of SEQ ID NO: 1 derived from the strain Brucella abortus , Dps (DNA starvation / stationary phase protection protein) consisting of the amino acid sequence of SEQ ID NO: 2, amino acid of SEQ ID NO: 3 A subunit vaccine composition for the prevention or treatment of brucellosis infection comprising AspC (aspartate aminotransferase) consisting of a sequence and a nucleoside diphosphate kinase (Ndk) protein consisting of the amino acid sequence of SEQ ID NO: 4 as an active ingredient. delete delete delete A method for the prevention or treatment of brucellosis infection comprising the step of administering the subunit vaccine (subunit vaccine) composition of claim 1 to a livestock that is expected to develop or develop brucellella infection. InpB (invasion protein B) consisting of the amino acid sequence of SEQ ID NO: 1 derived from the strain Brucella abortus , Dps (DNA starvation / stationary phase protection protein) consisting of the amino acid sequence of SEQ ID NO: 2, amino acid of SEQ ID NO: 3 A feed composition for preventing or improving brucellosis infection comprising AspC (aspartate aminotransferase) consisting of a sequence and Ndk (nucleoside diphosphate kinase) protein consisting of an amino acid sequence of SEQ ID NO: 4 as an active ingredient.

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