KR20140071131A

KR20140071131A - Vaccine composition comprising outer membrane protein of Salmonella Gallinarum for preventing fowl typhoid

Info

Publication number: KR20140071131A
Application number: KR1020120139130A
Authority: KR
Inventors: 한태욱; 한장혁; 장주현
Original assignee: 강원대학교산학협력단; 주식회사 고려비엔피
Priority date: 2012-12-03
Filing date: 2012-12-03
Publication date: 2014-06-11

Abstract

The present invention relates to a vaccine composition to prevent fowl typhoid containing an inactivated outer membrane protein of Salmonella gallinarum and an adjuvant as active ingredients, and to a method to prevent fowl typhoid using the same. According to the present invention, an outer membrane protein antigen of a Salmonella gallinarum field strain exhibits a very excellent effect of defending against Salmonella gallinarum even with only a single vaccination with a small content thereof, thus can be useful in preventing fowl typhoid.

Description

[0001] The present invention relates to a vaccine composition for preventing poultry typhus comprising an extracellular membrane protein of Salmonella gallina room,

The present invention relates to a vaccine composition for the prevention of poultry typhus comprising an inactivated intracellular protein and an adjuvant of Salmonella galina room as an active ingredient, and a method for preventing poultry typhus using the same.

Salmonella, the most important cause of human foodborne illness, was first reported by the veterinary bacteriologist Salmon in 1886, and has since been reported to be serotonergic in response to the serological response to O-antigen and H-antigen of LPS (lipopolysaccharide) To date, more than 2,500 serotypes have been reported. In this study, we investigated the effects of Salmonella spp. On the growth of Salmonella spp. In the wild, but the majority of Salmonella spp. It is presumed that the role of algae is important.

Among Salmonella Galina Room (Salmonella Gallinarum, SG) it will have played a steady occur sporadically since the country occurred in 1992 as a woninche causing the fowl typhoid causing economic losses to the poultry industry (Clarke and Gyles, 1993). The Salmonella Galina Room, which has host specificity, is highly pathogenic in chickens and suffers from deadly diseases with severe clinical symptoms. In general, Salmonella with host specificity In case of serotype, pathogen control and disease eradication is easier than serum type with broad host range. Salmonella galina room is infected with mammals, especially in poultry rodents, Due to the nature of the rodents, propagation due to this occurs well and the eradication is more difficult (Popoff et al ., 2004).

In order to eradicate the Salmonella Galina room, reports of sporadic occurrence of SG 9R vaccine and inactivated vaccine have been reported since the early 2000s (Babu et al ., 2003). However, SG 9R induces the immune response in the body. However, since the mechanism of attenuation of the live vaccine is not known, the possibility of restoration of the pathogenicity and the lesion in the liver or spleen in the host are reported, Or forbids use (Silva et al ., 1981; Smith, 1969; Wigley et al ., 2005). However, in many developing countries, including Korea, the outbreaks of Salmonella Galina Room are resistant to conventional antibiotics, limiting the use of antibiotics. Therefore, despite the above mentioned side effects, SG 9R is a preventive measure against poultry typhus And it is necessary to develop a new vaccine that is safe and safe to replace it.

Since the salmonella is generally protected by cell-mediated immunity on the proliferative nature of host cell lymphocytes and macrophages (Collins and Campbell, 1982; Lillehoj, 1991), vaccines using various methods are under study, , The attenuated live bacterial vaccine is more effective than the inactivated vaccine (Babu et < RTI ID = 0.0 > al ., 2003), there is a disadvantage that safety problems such as recovery of poisonous power and possibility of lesion in host may be raised.

On the other hand, outer membrane proteins (OMPs) of Salmonella have been reported as immunodominant proteins that activate cell mediated immunity (Lee et al al ., 2010; Meenakshi et al ., 1999), Bouzoubaa et al. (Bouzoubaa et al., 1987) demonstrated that the OMP antigen is superior to the SG 9R vaccine used as a live vaccine preparation for the prevention of poultry typhus.

Therefore, the present inventors have completed a method for massively isolating an OMP antigen, an extracellular membrane protein, from an outdoor strain of Salmonella Galina Room which infects poultry in Korean poultry and causes poultry typhus, and instead of inactivating the whole cell of Salmonella Galina Room, The vaccine was prepared with an adjuvant that can assist in the induction of cell mediated immunity with the OMP antigen of Salmonella Galina Room isolated by using.

The inventors of the present invention were studying the inactivated vaccine of Salmonella Galina Room causing poultry typhus, confirming that the vaccine containing the extracellular membrane protein antigen of Salmonella Galina Room outdoor strain can effectively prevent the infection of Salmonella Galina Room, Thereby completing the invention.

Accordingly, the present invention provides a vaccine composition for preventing poultry typhus comprising an extracellular membrane protein antigen of Salmonella Galina Room outdoor strain, and a method for preventing poultry typhus using the same.

The present invention provides a vaccine composition for the prevention of poultry typhus comprising an inactivated cell membrane protein and an adjuvant of Salmonella galina room as an active ingredient, and a method for preventing poultry typhus using the same.

The extracellular membrane protein antigen of the Salmonella Galina Room outdoor strain according to the present invention exhibits excellent effect of defending Salmonella Galina Room even when it is inoculated only once with a small dose, so that it can be effectively used for preventing pheosis typhus.

FIG. 1 is a diagram showing the staining (1A) and the immunoblot analysis (1B) after two-dimensional electrophoresis performed for the proteome analysis of the extracellular membrane protein of the Salmonella Galina Room of the present invention.

The present invention provides a vaccine composition for the prevention of poultry typhus comprising an inactivated cell membrane protein of Salmonella Galina Room and an adjuvant as an active ingredient.

In addition, the present invention provides a method for preventing poultry typhus comprising the step of inoculating the poultry with the vaccine composition for preventing poultice typhus.

Hereinafter, the present invention will be described in detail.

In the present invention, a vaccine composition for preventing poultry typhus is prepared by extracting extracellular proteins from Salmonella gallinarum (Salmonella gallinarum), which is a causative organism of poultry typhus causing economic damage in domestic and overseas poultry industry, and inactivating the extracellular proteins.

In the production of a vaccine for preventing poultice typhus, when the whole microorganism of the Salmonella Galina Room is inactivated, LPS (lipopolysaccharide), which acts as an endotoxin of Salmonella, is included and the swelling, Fever and loss of appetite, and it has been reported that the defensive ability is poor, and avoids the use of inactivated vaccine in the general farm. However, the inactivated vaccine prepared by extracting the outer membrane protein (OMP) of the Salmonella Galina Room according to the present invention is composed of only the extracellular proteins, and thus has excellent safety and defense.

The extracellular membrane protein according to the present invention is a constitutive protein related to the immune response as a result of two-dimensional electrophoresis, immunoblot analysis, and PMF (peptide mass fingerprinting) analysis. OMPA (outer membrane protein A), one of the three proteins, is known to stimulate the T lymphocyte response and cell-mediated immunity in the host, which is a gene that transcribes this protein (Cordwell, 2006; Lee et al ., 2007), a vaccine containing OMP A as an active ingredient of the inactivated cell outer membrane protein of the present invention can effectively prevent poultry typhus by stimulating the host's immune system.

The effects of inactivated protein vaccines using extracellular proteins have already been demonstrated in several papers, but the amount of protein reaching the immune response has been limited (Meenakshi et al ., 1999). In general, a high content of more than 400 μg has a disadvantage of showing a sufficient defense effect. However, the vaccine composition containing the extracellular membrane protein of the Salmonella Galina Room of the present invention as an active ingredient can be used as an effective ingredient in the vaccination of Salmonella Galina Room with a low content of 100% by using an effective adjuvant which induces humoral and cell mediated immune responses. Of survival rates and maintain a strong and sustained immune response.

The composition of the present invention may be at least one selected from the group consisting of ISA70 VG, ISA71 VG, ISA760 VG, ISA763A VG, ISA775 VG, and ISA780 VG, which are used as an adjuvant for the vaccine for poultry, But is not limited thereto.

The vaccine composition of the present invention is prepared by mixing a mixture of an inactivated cell membrane protein of Salmonella Galina Room with a phosphate buffer and an adjuvant. In this case, the mixing ratio of the mixture and the sorbent is 3: 7 to 5: 5 (v / v), preferably 4: 6 (v / v).

In the vaccine composition of the present invention, the daily dose of the Salmonella Galina Room extracellular protein antigen may be about 25 to 400 μg, preferably about 50 to 200 μg, depending on the clinical test results, Can be administered separately.

In the case of a vaccine containing an existing extracellular membrane protein as an active ingredient, the twice-inoculation has been reported to have a superior effect in the case of one inoculation (Bouzoubaa et al ., 1987), the vaccine composition of the present invention exhibits the same effect as that of the second inoculation even with only one inoculation, which is excellent in economics.

The vaccine composition of the present invention can be administered to birds by various routes. All modes of administration may be expected, for example, by intramuscular, intraperitoneal, intravenous, subcutaneous or intranasal routes, and preferably intramuscularly, but are not limited thereto.

In addition, the vaccine composition of the present invention may be administered alone or in combination with other vaccine compositions that prevent poultice typhus.

The poultry to which the vaccine composition of the present invention is applied may be a chicken, a duck, a turkey, a queen, a pigeon, a canaria, and the like, but is not limited thereto.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1. Salmonella Galina's Room Of the extracellular membrane protein (OMP) detach

1-1. Salmonella Galina Room Outdoors Selection and Culture

Candidates for vaccine The outbreaks of Salmonella Galina Room isolated from Wonju, Gangwon Province (2000), which showed A type in the PFGE type using restriction enzymes of Xba I and Spe I, are candidates for the outer membrane protein (OMP) vaccine Mainly selected (Seo et al ., 2006). The selected vaccine candidate strains were cultured in TSB liquid medium (Tryptic soy broth, BD Difco, USA) for 18 hours at 37 ° C, centrifuged at 13,000 rpm for 20 minutes at 4 ° C, Was used for protein extraction.

1-2. Salmonella Galina Room Outdoors Of the extracellular membrane protein (OMP) detach

Extraction of extracellular protein (OMP) from Salmonella Galina Room outdoor strain was simplified and the extraction method was simplified to reduce cost by separation method. The Salmonella Galina Room cells precipitated in 1-1 above were transferred to Macfarland No. 1. 10 and resuspended. The modified Barenkamp method (Barenkamp et al ., 1981), the microbial cells were pulverized using an ultrasonic grinder (SLTt, USA) and centrifuged at 8,000 rpm for 30 minutes using a centrifuge to remove only the supernatant containing OMP antigen Respectively.

Example 2. Salmonella Galina Room Extracellular membrane protein ( OMP ) Proteome analysis of antigen

2-1. Two-dimensional electrophoresis (2- Dimentional come 전공기 )

Modified Rabilloud's method (Rabilloud et al., 1998) isoelectric focusing (isoelectric focusing, IEF) to the IPG (immobilized pH gradient) in 7M urea concentration to strip (urea), thiourea (thiourea) of 2M concentration, the concentration of 2% CHAPS (3- according to A reswelling solution consisting of [(3-cholamidopropy) dimethyammonio] -1-propanesulfonate, DTT (dithiothreitol) at 1% concentration and pharmalyte at 1% concentration was subjected to reswelling Respectively. 200 μg of each sample was used per strip, and the IEF was performed at 20 ° C in accordance with the manufacturer's instructions using a Multiphore II system from Amersham Biosciences. The conditions of the IEF were set to reach the time from 150 V to 3,500 V for 3 hours and to last for 26 hours at 3,500 V to be finally set at 96 kVh.

Next, before performing SDS-PAGE (sodium dodecyl sulfate-olyacrylamide gel), the IPG strips were washed with equilibration buffer (50 mM Tris-Cl, pH 6.8, 6M urea, 2% % glycerol) for 10 min and immediately washed with equilibration buffer containing 2.5% iodoacetamide for 10 min. The equilibrated strips were arrayed on SDS-PAGE gels (20x24 cm, 10-16%) and developed to 1.7 kVh at 20 ° C using a Hoefer DALT 2D system (Amersham Biosciences, USA). Two - dimensional electrophoresis of the two - dimensional gel was performed and the proteins were spotted and spotted.

The results are shown in Figure 1A.

As shown in FIG. 1A, in the proteome analysis performed to determine whether any of the components of the extracellular membrane protein is related to the immunogenicity in the host, the isoelectric point of the extracellular membrane protein and the molecular weight of the extracted extracellular protein As a result of staining, 22 spots were observed, and most of the proteins were found to be distributed within the range of 10-15 kDa and 30-45 kDa and the isoelectric point within the range of 4-8.

2-2. Immunoblot analysis And PMF ( peptide mass fingerprinting ) analysis

Two-dimensional electrophoresis was carried out in the same manner as in 2-1 above. Proteins on SDS-PAGE gels subjected to two-dimensional electrophoresis for immunoblot analysis were transferred to a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech, USA). Immunoblot analysis was performed by modifying the method of Wu (Wu et al ., 2002).

First, the PVDF membrane was reacted at 37 ° C for 1 hour in a solution of TBS-T (50 mM Tris-Cl pH 7.5, 200 mM NaCl, 0.5% Tween 20) supplemented with 5% fetal bovine serum , And the brown antiserum obtained by inoculating Salmonella Galina Room vaccine twice at intervals of 2 weeks was diluted 1: 100 and reacted for 2 hours at 37 ° C. Then, anti-chicken IgG diluted at a ratio of 1: 1000 was reacted at 37 ° C for 1 hour. Finally, the antigen antibody conjugate was stained by reacting with DAB (3,3'-diaminobenzidine tetrahydrochloride). The reacted proteins were compared with the proteins visualized by staining in 2-1. Then, trypsin was treated to analyze the peptide mass fingerprinting (PMF) as a unique peak value of each protein obtained by mass spectrometry. And was identified as a unique protein by the amino acid sequence based on the NCBI database.

The results are shown in FIG. 1B and Table 1.

As shown in FIG. 1B, the reaction with Salmonella Galina Room antiserum for immunoblotting assay resulted in an antigen-antibody reaction in only 9 spots.

As shown in Table 1, PMF analysis was performed to identify 9 spots of the protein. As a result, all of the remaining 8 spots except for one spot were identified. Among them, OMP channel protein (OMP channel protein), which is a protein having four isoforms, has been identified most frequently. OMP A (outer membrane protein A), DNA starvation protein Protective proteins (stationary phase protection proteins) each had two isoforms.

Example 3. Salmonella Galina Room Extracellular membrane protein Preparation of vaccines containing antigens

Example 1 A cell outer membrane protein isolated from 0.3% (outer membrane protein, OMP) ( v / v) followed by the addition of formalin, was screen 3 days inactivated at 37 ° C BCA ^TM Protein Assay Kit (Thermo Scientific, USA ), The concentration of the protein was adjusted to 10,000 μg / mL, and the test vaccine was prepared under refrigeration.

A mixture of inoculated cell membrane protein antigens in phosphate buffered saline (PBS) in various amounts (6.3 μg, 12.5 μg, 25 μg, 50 μg, 100 μg and 200 μg) v / v) and mixed with Tissue Lyser II (Qiagen, USA) at 4,000 rpm for 20-30 minutes to prepare a vaccine containing the extracellular membrane protein antigen of Salmonella Galina Room.

Example 4. Salmonella Galina in the room Defense immunity test

The protective effect of the test vaccine and antibody-forming ability were investigated in 2-week-old brown laying hens which were highly susceptible to Salmonella Galina Room by 1) concentration of antigen, 2) inoculation route, and 3) number of inoculation. 1) The concentration of antigens was 5, 200, 100, 50, 25, 12.5, 6.3 μg in 5 groups, and 2 weeks after the inoculation, 2) Inoculation route was intramuscular injection and subcutaneous injection I divided it. 3) The number of inoculations was divided into 1 vaccination group and 2 vaccination groups, and 2 weeks after the vaccination was completed, attack was inoculated.

4-1. Extracellular membrane protein ( OMP ) Salmonella according to the concentration of antigen Galina in the room Protective Effect and Antibody Formability

In order to determine the effective amount of the vaccine containing the extracellular membrane protein of the Salmonella Galina Room of the present invention as an active ingredient, 200, 100, 50, and 50% of the vaccine group was divided into five groups, 25, 12.5, and 6.3 μg / dose were administered to the thoracic muscle. Two weeks after the vaccination, all survivors were vaccinated and observed for 2 weeks.

The results are shown in Table 2.

As shown in Table 2, the vaccine-free survival rate showed a survival rate of 20%. In contrast, the survival rate was 100% in the vaccination group inoculated with 25 μg / dose of the extracellular membrane protein, The efficacy of the vaccine has been demonstrated. However, the results of observation of pathologic tissues after 2 weeks of the inoculation showed that the vaccine containing 25 μg of extracellular protein antigen showed severe nodules and hyperplasia of the heart, hyperemia, We could confirm the changed gross lesion. However, since only a slight extent of the liver was observed at over 50 μg, the extracellular protein antigen amount of Salmonella Galina room of 50 μg or more shows sufficient protection and safety of poultice typhus in the host's body.

4-2. Extracellular membrane protein ( OMP ) Of the vaccine On the inoculation route Salmonella according to Galina in the room Protective Effect and Antibody Formability

The following experiment was conducted in order to minimize the side effects of the vaccine containing the extracellular membrane protein of the present invention as an active ingredient and to find the most suitable route for inducing the effect of the vaccine.

First, the control group was divided into five groups, namely muscle group and subcutaneous group. Muscle administration group was inoculated to brown thoracic muscle, and pia group was subcutaneously inoculated. Each group was divided into 200, 100, 50, and 25 μg by the dose of the extracellular protein antigen, and the survival rate and the adequacy of each route were reevaluated.

The results are shown in Table 3.

As shown in Table 3, the survival rate of all the extracellular membrane protein antigens was 100% at the time of inoculation, whereas at the time of subcutaneous inoculation, the survival rate was 100% only at 200 and 100 μg, and in the case of 50 μg and 25 μg 80% and 60%, respectively.

Therefore, it was confirmed that the muscle inoculation route is the most effective route of administration in terms of survival rate. In the gross lesions, subcutaneous inoculation showed epicarditis and heart nodule at 20 μg, and severe subcutaneous liver and spleen enlargement was observed in subcutaneous inoculation route than subcutaneous inoculation even at the submaximal extracellular protein antigen concentration.

4-3. Extracellular membrane protein ( OMP ) Salmonella by vaccination frequency Galina in the room Protective Effect and Antibody Formability

In order to evaluate the protective effect of the vaccine containing the extracellular membrane protein of the present invention as an active ingredient, the number of 35 laying hens was divided into seven groups of five, each divided into one vaccination group and two vaccination groups. The duration between the first vaccination and the second vaccination of the second vaccination group was 3 weeks, the administration route was administered to the thoracic muscle, and the extracellular protein antigen dose was 200, 100, and 50 μg, respectively.

The results are shown in Table 4.

As shown in Table 4, survival rates of 100% of the respective extracellular protein proteins were shown in both the one vaccination group and the second vaccination group. In addition, the difference in gross lesions was observed in the 200 μg administration group of the once vaccinated group, and the slight change in the liver and yellow color was also observed in the same dose group of the twice vaccination group, But the efficacy and the response in the host were not significantly different.

Therefore, the vaccine containing the extracellular membrane protein of the present invention as an active ingredient exhibits the same effect as the inoculation of two or more times even by one inoculation, which is very economical.

Claims

A vaccine composition for preventing poultry typhus comprising an inactivated cell membrane protein of Salmonella gallinarum and an adjuvant as an active ingredient.

The vaccine composition according to claim 1, wherein the poultry is at least one selected from the group consisting of chickens, ducks, turkeys, quails, pigeons, and canaries.

The vaccine composition according to claim 1, wherein the adjuvant is at least one selected from the group consisting of ISA70 VG, ISA71 VG, ISA760 VG, ISA763A VG, ISA775 VG, and ISA780 VG.

The method according to claim 1, wherein the inoculated cell membrane protein of the Salmonella Galina Room is mixed with a phosphate buffered saline and the mixing ratio of the mixture and the ejuvant is 3: 7 to 5: 5 (v / v) Wherein the vaccine composition is a vaccine composition.

A method for preventing poultry typhus in a poultry, comprising the step of inoculating the poultry with the vaccine composition of any one of claims 1 to 4.

6. The method according to claim 5, wherein the poultry is selected from the group consisting of chickens, ducks, turkeys, quails, pigeons and canaries.

6. The method according to claim 5, wherein the inoculation route of the vaccine composition is at least one selected from the group consisting of intramuscular, intraperitoneal, intravenous, subcutaneous, or nasal passages.