KR101774863B1 - Salmonella enteritidis mutant strains hid2092 and hid2114 and pharmaceutical composition using the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating Salmonella enteritidis mutant and a salmonellosis including the mutant, a feed additive including the Salmonella enteritidis mutant strain and a mutant strain of Salmonella enteritidis to prevent and prevent salmonellosis, ≪ / RTI > The strain of Salmonella enteritidis according to the present invention provides a stable live bacterial vaccine which is safe and has stable genetic traits and high defense efficacy against salmonellosis, by preventing the recovery of virulence of attenuated strains. In addition, the Salmonella enteritidis mutant strains can be used for the production of a vaccine composition or feed, and salmonellosis of humans and animals (especially birds) can be effectively prevented and treated by using the vaccine composition or the feed composition.

Description

[0001] The present invention relates to mutant strains HID2092 and HID2114 for the treatment of salmonellosis, and salmonellosis pharmaceutical compositions containing the same. BACKGROUND ART [0002]

The present invention provides a pharmaceutical composition for preventing and treating salmonellosis, comprising Salmonella enteritidis mutant strains HID2092 and HID2114, a feed composition comprising the Salmonella enteritidis mutant strains HID2092 and HID2114, and Salmonella enteritidis mutant strains HID2092 and HID2114 To an animal to prevent and treat salmonellosis.

Salmonellosis is a common human and poultry disease that occurs when foods contaminated with bacteria are ingested. Symptoms of acute gastroenteritis occur after 12 to 48 hours of ingestion of contaminated food, and in cases of diarrhea, abdominal pain, vomiting and fever It also leads to death by sepsis.

Human Salmonella infections are classified into two types: typhoidal salmonellosis and non-typhoidal salmonellosis. Typhoid-type salmonellosis is a major problem when insufficient hygienic management occurs due to the infection of Salmonella Typhi bacteria through human-infected foods. However, non-typhoidal salmonellosis is a serious problem in developed countries It is also known as typhoid fever, and it is a major threat to public health in the countries of advanced countries such as the United States and Europe that have overcome typhoid fever type salmonellosis in relation to changes in livestock industry and processing and preservation of livestock products. In Korea, Salmonella laminaria and Salmonella enteritidis have caused serious problems in the Korean poultry industry for several decades and food poisoning caused by salmonella causing annual salmonellosis of non-typhoid type has been occurred.

It is known that S. enteritidis is the most common serotypes in non-typhoid salmonellosis of people with developed country-type salmonellosis, although there are differences in regions by country. Therefore, Practical application is urgently required.

Salmonella enteritidis vaccines, which have been or are being studied until recently, can be largely classified as inactivated vaccines, subunit vaccines, and live attenuated vaccines. Inactivated vaccine has safety advantages because it does not return to virulent pathogenicity. However, it induces mainly liquid immunity and induces cell - mediated immunity, which is significantly lower than that of live vaccine. Subunit vaccines, like inactivated vaccines, are more stable than live vaccines, but their efficacy is relatively short and the vaccination is required several times. Compared to the other two vaccines, live vaccine has various advantages such as diversity of inoculation method, humoral immunity, mucosal immunity and cell mediated immunity, and also can carry heterologous antigen.

On the other hand, there are about three live vaccines currently in practical use. ① There are Megan Vac ™ 1 (Megan Health, Inc.) using the attenuated S. Typhimurium △ △ cya crp strains. But it is pointed out that the defensive power is not enough. This is due to the use of salmonella typhimurium as a vaccine strain, as well as the metabolism-related genes cya And crp This is probably because the gene is missing. cya and crp When a gene is deficient, the bacteria lose their ability to ferment saccharides including maltose, and their viability is markedly degraded. Also, SG9R (poultice typhus vaccine, Intervet / Schering-Plow Animal Health) may be used to prevent S. enteritidis. However, even the same Salmonella strains have different disadvantages in that their serotypes are different from their serotypes if they have different serotypes, and there is a possibility of restoring the pathogenicity to S-type. ③ S. Enteritidis and S. There are TAD Salmonella vac E and T (Lohmann AnimalHealth, Germany) using chemically mutated strains of Typhimurium, but this is a genetically unspecified variant strain and has safety and stability problems.

In other words, there is almost no commercially available live vaccine showing definite protective effect and immunogenicity. Therefore, it is urgently required to develop a live bacterial vaccine which is safe and has stable genetic trait and sufficient defense force by preventing restoration of pathogenicity of attenuated strains .

1. Korean Patent Publication No. 10-2012-0129777 2. Korean Patent Publication No. 10-2011-0126227 3. Korean Patent Publication No. 10-2012-0128583

The present invention provides a live bacterial vaccine which is safe and has stable genetic traits and is excellent in preventing and treating salmonellosis by preventing the recovery of pathogenicity of attenuated strains.

In order to achieve the above object, the present invention rfaH gene in the chromosome of Salmonella bacteria Entebbe utility disk or msbB Thereby providing a mutant strain of Salmonella enteritidis in which the gene is inactivated.

In one embodiment of the present invention, inactivation of the gene may be by deletion of part or all of the gene of interest.

In one embodiment of the invention, rfaH The gene is composed of the nucleotide sequence of SEQ ID NO: 1, msbB The gene may be composed of the nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the invention, rfaH The Salmonella enteritidis mutant strain in which the gene is inactivated is a strain of Accession No. KCTC 12797BP, msbB The strain-inactivated Salmonella enteritidis strain may be a strain of the accession number KCTC 12798BP.

The present invention also provides a pharmaceutical composition for preventing or treating salmonellosis, which comprises the mutant Salmonella enteritidis strain of the present invention as an active ingredient.

In one embodiment of the present invention, the composition may be a live bacterial vaccine composition.

Also, the present invention provides a method for preventing or treating salmonellosis by inoculating an algae with a mutant Salmonella enteritidis strain of the present invention.

The strains of Salmonella enteritidis according to the present invention prevent the restoration of the virulence of the attenuated strains, thereby providing a stable live vaccine composition having stable genetic traits and excellent salmonellosis prevention and treatment efficacy. In addition, the Salmonella enteritidis mutant strain can be used for the production of a vaccine composition or feed, and the vaccine composition or feed can be used to effectively prevent and treat salmonellosis in humans and animals.

Fig. 1 shows the result of confirmation experiment of LPS mutant strains prepared using the lambda red recombination system. Figure 1a rfaH gene-deficient strain (Example 1, Δ rfaH), Figure 1b is a msbB The results for gene-deficient strain (Example 2, Δ msbB) (Lane M : DNA marker)
Fig. 2 shows the results of a culture experiment of a mutant strain according to the present invention. FIG. 2A shows the results of the experiments in the LB medium, and FIG. 2B shows the results of the experiments in the M9 minimal medium. Comparative Example (wild strain) -wild type, Example 1-? RfaH , Example 2- ? MsbB .
Fig. 3 shows the results of an experiment measuring the degree of LPS expression (pattern). Lane 1 - (wild strain), lane 2 - Example 1 ( ΔrfaH ), lane 3 - Example 2 ( Δ msbB ).
Figure 4 shows the results of an experiment evaluating the survival rate of animals. (Wild strain) -wild type 1403, Example 1-? RfaH , Example 2- ? MsbB , (*** significant difference compared with wild-type strain, p <0.001)
Fig. 5 shows experimental results (3 days and 14 days after inoculation) in which colonization was measured. 5A is a liver, and FIG. 5B is a spleen. Comparative Example (wild strain) -wild type, Example 1- ? RfaH , Example 2- ? MsbB (** p < 0.01, *** p < 0.001. In comparison with wild type strain on the third day after inoculation, there was a significant difference in mutant strains, and &'Φ' is compared with 3 days of inoculation and 14 days of inoculation, 'ND' can not be measured due to death of an individual)
FIG. 6A shows a liver weight ratio to a body weight, and FIG. 6B shows a body weight to weight ratio. _{(*** p &lt; 0.001.)}
7 is a photograph showing the spleen of each experimental group. Control-control, Comparative Example (wild-type strain) -SE 1403, Example 1- ? RfaH , Example 2- ? MsbB .

In order to accomplish the above object, the present invention provides a Salmonella Enteritidis mutant strain in which the rfaH gene or msbB gene is inactivated in the chromosome of Salmonella enteritidis, a pharmaceutical composition for preventing and treating salmonellosis comprising the same , A feed composition for prevention and treatment of salmonellosis containing the same, and a method for preventing and treating salmonellosis using the same. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to the accompanying drawings.

The present invention is characterized by a Salmonella enteritidis mutant strain in which the rfaH gene or the msbB gene is inactivated on the chromosome of Salmonella enteritidis.

Salmonella enteritidis is a causative organism of bacterial infection-type food poisoning in Salmonella spp. This bacterium is a representative bacterium of Salmonella and can cause infection in all animals and has very high host adaptability. Gram negative anaerobic bacterium with motivated (周 毛) is a mobility. It has no lactose decomposing power, does not form indole, does not produce hydrogen sulfide, and is distinguishable from E. coli. The optimal temperature for growth is 35 ~ 37 ℃, the temperature range for growth is 10 ~ 43 ℃ and it is killed by heating at 60 ℃ for 20 minutes. The optimum pH is 7.2-7.4. The size is 0.5 ~ 0.8 × 3 ~ 4μm.

The rfaH gene or msbB gene is a gene associated with Salmonella lipopolysaccharide (LPS). The Salmonella lipopolysaccharide plays an important role in adhesion of the host cell surface, intestinal colony formation, serum resistance, intracellular replication, and the like. O-antigen polysaccharide, core oligosaccharide, And lipid A (lipid A).

The rfaH RfaH expressed by the gene controls the biosynthesis of O-antigen polysaccharides and central oligosaccharides as transcription factors and is involved in the cell survival of bacteria and protects bacteria in harsh environments. The msbB MsbB, expressed by the gene, acts as an acyltransferase to supplement myristic acid to the lipid A precursor.

The Salmonella enteritidis mutant strain used in the present invention includes an inactivated rfaH gene or an inactivated msbB gene. According to one embodiment of the present invention, inactivation of the gene is caused by deletion of the gene of interest.

The gene of rfaH inactivated, preferably deficient for the production of the mutant strain according to the present invention has a nucleotide sequence shown in SEQ ID NO: 1, msbB The gene has a nucleotide sequence shown in SEQ ID NO: 2.

In general, inactivation of a gene group in a strain can be made in various forms. For example, modification of the signal structure of gene expression or antisense-RNA technology can reduce gene expression. For example, the signal structure of a gene expression may include, for example, a repressor gene, an activator gene, an operator, a promoter, an attenuator, a ribosome binding site, a start codon, Terminators include but are not limited to Jensen and Hammer (Biotechnology and Bioengineering 58: 191 195 (1998)), Carrier and Keasling (Biotechnology Progress 15: 58 64 (1999)), Franch and Gerdes Microbiology 3: 159 164 (2000)) and Knippers ("Molecular Genetics", 6th edition, 1995) or Winnacker ("Genes and Clones", 1990).

In addition, double-stranded RNA (dsRNA) composed of a sense strand having a sequence homologous to the mRNA of the target gene and an antisense strand having a sequence complementary thereto is introduced to inhibit the expression of the target gene by inducing degradation of the mRNA of the target gene RNA interference (RNAi) method, which is a mechanism that allows the use of the RNA interference method. Alternatively, it is possible to use a method of mutating via a transposon, which is a DNA sequence capable of moving to another position in the genome of a single cell, to inactivate the function of the target gene by blocking the function of the target gene. Mutations that induce a change or decrease in catalytic activity of a gene family protein are well known in the art (Qiu and Goodman, Journal of Biological Chemistry 272: 8611 8617 (1997)), Yano et al. (Proceedings of the National Academy of Sciences Science, USA 95: 5511 5515 (1998)), Wente and Schachmann (Journal of Biological Chemistry 266: 20833 20839 (1991), and Hagemann ("General Genetics", 1986).

Preferably, inactivation of the gene group may be accomplished by mutation of single or multiple base sequences, deletion of a single or complex gene, insertion of a foreign gene into the gene, deletion of the whole gene group, insertion of the inhibition sequence of the promoter of the gene group, , Insertion of regulatory inhibition of gene expression, insertion of RNAi into single or multiple base sequences of the gene family, transposon mediated mutation, or a combination of these variants.

The methods described above are generally understood by those of ordinary skill in the art and can be performed as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Press 2001) .

The activity of a protein expressed from a gene may be greatly reduced by changing a single or a complex sequence of the active site in the base sequence of the gene, or a deletion of a single or complex gene, an antibiotic resistance gene or a foreign gene So that the complete protein can not be expressed. Preferably, a method of deleting all the nucleotide sequences of genes present on the chromosome can be used (Russell CB et al., J. Bacteriol. (1989); 171: 2609-2613, Hamilton CM et al, J. Bacteriol. 1989); 171: 4617-4622, Link AJ et al., J. Bacteriol. (1997); 179: 6228-6237). Or a combination of mutants by the above method.

In the embodiment of the present invention, a method of deficient all the base sequences in the chromosome, which is the last method, is used to most inactivate the pathogenic gene. The method of deficiency of the gene group is called 'Onestep deletion method' (Datsenko KA et al., PNAS) using a lambda red recombinase developed by Datsenko KA et al., Widely known as an E. coli gene deletion method , (2000); 97 (12): 6640-6645).

According to one aspect of the present invention, there can be provided a pharmaceutical composition for preventing or treating salmonellosis including a mutant strain of Salmonella enteritidis, particularly a live vaccine composition.

The present inventors deposited the Salmonella enteritidis mutant strains prepared in the present invention to a depository and assigned accession numbers. The mutant strains in which the rfaH gene was deleted were named as "HID2092" and 2015 The present inventors named the strain as "HID2114" for the mutant strain in which the gene of msbB was deleted, and the name of the strain was named "HID2114" in 2015 On April 21, the strain was donated to the Korea Research Institute of Bioscience and received the deposit number as "KCTC 12798BP".

The term &quot; prevention &quot; in the specification of the present invention means inhibiting the occurrence of diseases or diseases in an animal which has never been diagnosed as having a disease or disease, but which tends to be susceptible to Salmonella disease or disease.

The pharmaceutical composition or vaccine of the present invention may further comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound.

The term "vaccine" in the present invention refers to a medicament that weakens an antigen, that is, a pathogen, and forms an antibody by injecting it into a human body to have resistance and immunity against the disease. There is a live vaccine that is poisoned. In the present invention, the mutant Salmonella enteritidis strain is useful as a live vaccine for preventing Salmonella because it is a strain that has the same antigenic protein as the wild type strain and has a very low pathogenicity.

Examples of formulations for oral administration comprising the composition of the present invention as an active ingredient include tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs can do. A binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrating agent such as corn starch or sweet potato starch, a disintegrant such as magnesium stearate , Calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned materials, a liquid carrier such as a fatty oil may be further contained.

The parenteral dosage form containing the composition of the present invention as an active ingredient can be formulated into a main-use form such as subcutaneous injection, intravenous injection or intramuscular injection. For formulation into injectable formulations, the compositions of the present invention may be formulated as solutions or suspensions in water with stabilizers or buffers in water, and formulated for unitary administration of ampoules or vials.

The vaccine composition according to the present invention prevents salmonellosis of birds by preventing avian paratyphi, thereby preventing human salmonellosis in humans and preventing human food poisoning. The alga that is an object of the present invention may be a chicken, a duck, a turkey, a queen, a waterfowl, a dove, a canary, and the like, but is not limited thereto.

According to one aspect of the present invention, a feed additive is more preferably used than a livestock feed composition comprising a Salmonella enteritidis mutant strain. It acts as a feed additive and acts as a live vaccine to prevent Salmonella.

The feed composition of the present invention can be prepared by separately preparing a Salmonella enteritidis mutant strain in the form of a feed additive and mixing it with the feed or directly adding the feed to the feed. The strain of Salmonella enteritidis in the feed of the present invention may be in a liquid or dry state, and the drying method may be, but not limited to, air drying, natural drying, spray drying and freeze drying. In addition, the feed composition may further include conventional additives that can enhance the preservation of feed in addition to the Salmonella enteritidis mutant strains of the present invention.

The feedstuffs containing Salmonella enteritidis mutant strains in the present invention may include cereals, muscle roots, food processing busines, birds, fiber oils, pharmaceuticals buses, oils, fats, Examples of the animal species include, but are not limited to, proteins, inorganic substances, fats, mineral oils, single cell proteins, animal planktones, and residual foods.

Examples of the feed additives including the mutant Salmonella enteritidis strain according to the present invention include binders, emulsifiers, preservatives and the like added to prevent quality deterioration. In order to increase the efficiency, amino acids, vitamins, enzymes A preservative, a flavoring agent, a silkworm, a silicate, a buffer, a coloring agent, an extracting agent, an oligosaccharide, and the like, and may further include a feed mixture, but the present invention is not limited thereto.

According to one aspect of the present invention, there is provided a method of preventing or treating salmonellosis by inoculating an animal with a mutant strain of Salmonella enteritidis.

The composition of the present invention may be administered to an animal in the form of a pharmaceutical preparation or mixed with feed or water of livestock and fed to the animal, preferably mixed with the feed in the form of a feed additive .

The route of administration of the composition of the present invention can be carried out through various routes including parenteral, intraarterial, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal routes of administration, Lt; / RTI &gt;

A method of treatment of the invention comprises administering a composition of the present invention in a pharmaceutically effective amount. It will be apparent to those skilled in the art that the appropriate dosage can be determined by the practitioner within the scope of sound medical judgment. Specific therapeutic effective amounts will vary depending on factors such as the type and degree of response to be achieved, age, body weight, health status, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, And other similar factors well known in the medical field. However, it is generally possible to administer the drug in an amount of 10 ⁴ to 10 ⁸ CFU per one to several times.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example  1. Salmonella Entertidis  Mutant strain production

(1) Experimental strain and culture conditions

Salmonella enterica Enteritidis IVKW 1403 was obtained from Korean chickens and used in wild type in all experiments. Bacteria were cultured in Luria-Bertani (LB) broth or agar medium at 37 ° C and screening was performed using antibiotics (ampicillin 200 μg / ml, kanamycin 83 μg / ml, or tetracycline 35 μg / ml).

Salmonella strains of the Datsenko KA, such as the development of a lambda Red recombinase (lamda Red recombinase);: rfaH using (Datsenko KA et al, PNAS, (2000) 97 (12) 6640-6645) Gene and msbB A mutant strain in which the gene was deleted was prepared. The plasmids used for the production of the mutant strains are shown in Table 1. For reference, the rfaH The gene is the nucleotide sequence of SEQ ID NO: 1, and the msbB The gene is shown in the nucleotide sequence of SEQ ID NO: 2.

name characteristic pTP 233 Lambda red recombinase expression vector, ancillary plasmid, temperature sensitive ori; Amp ^R Tet ^R pKD4 FLP recognition sites (FRT) ((oriR, the template plasmid for amplification of the kan cassette side screen by Amp ^R Km ^R) pCP 20 temperature-sensitive plasmid (replicated at 30 캜) containing the flp recombinase gene, Cm ^R Amp ^R gene,

The primers were then used to amplify the kanamycin resistance gene of plasmid pKD4. Each PCR fragment was then inserted into the cell using electrophoresis. The cells were cultured in an LB agar medium containing kanamycin (83 mu g / ml) to select mutant strains. The selected mutant strain was confirmed using a suitable primer. The primers used in the above are shown in Table 2.

primer The sequence (5 '- &gt; 3') Msbb_long_F 0228 ATGGCGGGGATTGCATTAACACCGGCATCATTCCGTGACCCTTTGTAGGCTGGAGCTGCTTC Msbb_long_R 0228 TTATACGGCTGAATCTCGCCTGGCTTGCGGGTTTTGAGCAGCTTCCTCCTTAGTTCCTATTCCG Long_ rfaH _F CTAAATCTTGCGAAAACCGGTGTTTTTTACGCTCTGCTTCTGTAGGCTGGAGCTGCTTCG Long_ rfaH _R GCGGATAAGAGTCATTATGCAATCCTGGTATTTACTGTACCATATGAATATCCTCCTTAG MsbB_com_F2 CGGGCGCTAATTAATCTGTCT MsbB_com_R3 CGGTTCAACCAATACCACGCGTAT RfaH_com_F GATCTTACGCCGAAACCAA RfaH_com_R CATTGTAGCCTCTTGGTAAGC

Plasmid pCP20 was then used to remove the kanamycin resistance region.

(2) Identification of mutant strains

The rfaH produced according to the above- Gene deletion strains ( ? RfaH ), msbB Gene deletion strains ( Δ msbB ), rfaH Gene and msbB Genetic deletion strain ( Δ rfaH Δ msbB ) was confirmed to be a mutant strain by a colony PCR method (see FIG. 1).

division Explanation Comparative Example Salmonella entericapteristis IVKW 1403 wild strain Example 1 rfaH Gene deletion strain ( Δ rfaH ) Example 2 msbB Gene deletion strain ( Δ msbB )

The results of confirmation for Example 1 are shown in FIG. 1 (a), and the results for Example 2 are shown in FIG. 1 (b).

1, lane 1 shows a comparison example (wild strain), lane 2 shows a mutation type in which each gene is substituted with a kanamycin cassette, lane 3 shows a mutation form in which a kanamycin cassette is removed Yes). Variable strains of the gene size expected from all the mutant strains can be identified.

Specifically, in FIG. 1 (a), Lane 1: wild type, 815 bp; lane 2: RfHA :: KM, 1886 bp; lane 3: RFhH , 459 bp. 1 &lt; / RTI &gt; (b), Lane 1: wild type, 1141 bp; lane 2: msbB :: KM, 1845 bp; lane 3: msbB , 418 bp.

The mutant strains were deposited at the Korea Biotechnology Center. The mutant strains in which the rfaH gene was deleted were named as "HID2092". On April 21, 2015, The present inventors named the mutant strain "HID2114" for the mutant strain in which the msbB gene was deleted and deposited the strain as KCTC 12798BP on Apr. 21, 2015 at the Korea Research Institute of Bioscience and Biotechnology as "KCTC 12797BP"".

Example  2. Cultivation and growth of mutant strains

The mutant strains prepared above were cultured in LB medium and M9 minimal medium, respectively, and the degree of growth was evaluated. 5 ml of the mutant strain was inoculated into the LB medium, cultured for 17 to 18 hours (absorbance at 600 nm, 0.8), and transferred to 100 ml of the M9 minimal medium or LB medium, respectively. Bacterial growth was then measured at 2 hour intervals for 48 hours. The results of the above experiment are shown in Fig. 2 (Fig. 2 (a) shows the result of culturing in LB medium and Fig. 2 (b) shows the result of culturing in M9 minimal medium).

As a result of culturing in LB medium, all of the mutant strains did not grow until 2 hours after the incubation, and began to grow after 2 hours. The comparative examples (wild strains) showed the best growth, followed by growth in the order of Example 1, Example 2 and Example, but there was no significant difference in their growth rates. This means that the growth of the strain is not delayed due to the mutation according to the present invention.

As a result of culturing in the M9 minimal medium, the growth of all the strains was delayed at 6 hours of culture, and after 6 hours of cultivation, the growth was good except for the embodiment of the present invention. The growth of the example of the present invention was confirmed by culturing for 10 hours or more in the M9 minimal medium.

Also, when cultured in LB medium, the lag phase of all the mutant strains was terminated 6 hours earlier than when cultured in M9 minimal medium.

Example  3. LPS  Expression trend analysis

The mutant strains prepared by the above-described method were evaluated for the degree of LPS expression (trend) by electrophoresis and silver staining using 16% deoxycholate-polyacrylamide gel (DOC-page gel) Respectively.

Lane 2 shows the results of Example 1 ( Δ rfaH ), no upper portion of the LPS gene was shown, and the sample moved the fastest. This means that the 0-antigen chain is not synthesized in the mutant strains deficient in rfaH (Example 1).

Example 2 ( ΔmsbB ), which is another mutant strain, showed a similar tendency as compared with the wild type strain except that the lower inner central band exhibited a higher expression level than the comparative example (wild strain), but the expression level was significantly Respectively.

Example  4. Survival rate of salmonella-infected rats (virulence)

Seven weeks old BALB / c female mice were prepared, and the prepared mutant strains were injected into the abdominal cavity of mice at different concentrations, and the virulence was measured. Bacterial cells were washed twice and then serially diluted in 10X PBS solution. The inoculum of the mutant strains were different from each other by 10 ⁶ , 10 ⁷ and 10 ⁸ CFU / 100 μl (see FIGS. 4a, 4b and 4c).

Each group of mice with specific pathogen free (SPF) mice in 4 mice (control and 3 mice) was observed for 2 weeks post infection and recorded daily mortality. For the ethical use of experimental animals, the 3R principle was observed and an animal experiment was conducted under the regulation of the Animal Experiment Ethics Committee of Kangwon National University (Permission No. KW-130829-1).

The results of the experiment are shown in FIG. All mice inoculated with the comparative examples (wild strains) were killed on the third day after inoculation irrespective of the dose. In contrast, mice of the 10 ⁶ CFU / 100 ㎕ inoculated group of the present invention survived throughout the entire experimental period, and 50% of the mice in the 10 ⁷ CFU / 100 ㎕ inoculated group survived.

In the mice inoculated with 10 ⁶ CFU / 100 실시 of the examples, 100% of the subjects died on the third day of all groups inoculated with Example 1, whereas 50% of the mice in Example 2 survived on the whole of the experiment Respectively.

Overall, the survival rate of the inoculated group was significantly higher than that of the control group, and that the survival rate of the inoculated group was higher than that of Example 2 according to the present invention.

Example  5. Salmonella mutant strains Colony  formation

BALB / c female mice at 7 weeks of age were used to measure the colony formation of mutant strains. A group of 5 specific pathogen-free (SPF) mice were intraperitoneally injected with 10 ⁶ CFU / 100 μl of each mutant strain.

The animals were killed on days 3 and 14 after infection. After liver and spleen homogenization, bacterial colonies were counted in Salmonella- Shigella (SS) agar (BD, USA).

The results of the experiment are shown in FIG. On the third day after inoculation, bacterial colonies were 4.6 log CFU / g and 4.9 log CFU / g, respectively, in the liver compared to the wild-type strain (Comparative Example) in both the inoculated mutant strains (Example 1 and Example 2) g (see Fig. 5A).

The number of bacteria was not measurable on the 14th day after inoculation because all individuals in the inoculated group died before 14 days of inoculation. On the 14th day after inoculation, the mutant strain (Example 1 and Example 2) log CFU / g, 1.5 log CFU / g, respectively. In all the groups inoculated with the mutant strains, colony formation was significantly decreased on the 14th day after inoculation compared to the third inoculation.

On the third day after inoculation, the number of colonies of the group administered with Example 1 and Example 2 of the present invention was 6.3 log CFU / g and 6.8 log CFU / g, respectively, as compared with the wild-type group, statistically significantly 5B).

At 14 days after inoculation, the number of colonies formed in Example 2 in the spleen was significantly lower than that in Example 1. In addition, the colonization number of the inoculated group of Example 2 was significantly decreased at 14 days after inoculation compared to that of the inoculation group of 3 days.

On the third day of inoculation, the organ / body weight ratios of the experimental individuals were measured and shown in Figures 6a and 6b. The ratio of liver and spleen to body weight of all the groups inoculated with mutant strains (Examples 1 and 2) was significantly lower than that of wild-type inoculated group.

The body weight to liver ratio and the body weight to spleen ratio of the group administered with Example 2 of the present invention were the lowest, and the ratio was almost the same as that of the control group in which the germs were not inoculated.

< Example  6>

From the mouse Live vaccine Candidate Week  Korean defense rate test

The present inventors used 7-week-old BALB / c female mice as an experimental animal for IVF test for the live vaccine candidates in the mice, and were raised in IVC Rack (MVCS; Threeshine, Korea). In the case of the defense rate test, 4 weeks after the vaccination, 10 ⁸ CFU / mouse of Salmonella enteritidis HID1403 was inoculated into the mouse mouth, and the mice were weighed on the 7th day, and the number of attack bacteria detected from liver and spleen was measured Respectively. The isolated liver and spleen were diluted with PBS, and the tissue was disrupted with Tissue Lyser (QIAGEN, USA). The number of colonies was measured in Salmonella-Shigella (SS) agar (BD, USA). SE mutants were inoculated intraperitoneally (IP) titers in order to measure the protective effects of the mice. Phosphate-buffered saline (PBS) alone was administered to the negative control.

After 1 week of inoculation, all mice were killed and the liver and spleen were extracted. The number of colonies of the attack strain was measured and compared with the control group (2.8 × 10 ⁵ CFU / g), all groups were statistically significant ( P <0.001), respectively. This shows that the vaccine of the candidate vaccine is excellent. Compared with the control group (1.8 × 10 ⁶ CFU / g), the vaccine group had a significantly lower number of colonies ( p <0.001). No statistical significance was observed in the comparison between the embodiments (see FIG. 6).

< Example  7>

From the armor test mice Live vaccine Candidate Week  Antibody against

After 1, 2, 3, and 4 weeks of vaccination, blood was collected from orbital blood collection in all mice. ELISA was performed to identify changes in blood antibody. Each well was coated with 50 μl / well of antigen (Salmonella Enteritidis OMP or LPS, 10 / ml) and the plate (Nunc, Wiesbaden, Germany) was stored at -4 ° C for 16 hours. The plates were then washed three times with PBST (0.05% Tween 20 + PBS) and then blocked with 2% BSA (Merck, Darmsdadt, W. Germany) at 37 ° C. After washing again with PBST, 100-fold diluted serum was added to PBS containing 0.5% BSA as the primary antibody and incubated at 37 ° C for 1.1 hours. IgG, IgG1, IgG2a and IgA diluted 700 and 2500 times with the secondary antibody were added and cultured at 37 ° C for 1 hour. 100 TMB (SurModics, Eden Prairie, USA) was added and reacted for 3 minutes in the dark, then the reaction was stopped with 2N H ₂ SO ₄ and the absorbance was measured with an ELISA reader.

IgG1 (see FIG. 8B) and IgG2a (see FIG. 8C) were measured using whole cells as the antigens. As a result, the IgG1 And IgG2a were significantly higher in all groups except for IgG2a. Vaccination In 4 weeks, IgG, IgG1 showed significantly higher antibody titers in the vaccinated group than in the Example 1 group. In addition, the antibody level of Example 2 gradually increased during the experimental period from the first week of vaccination, while the antibody level of Example 1 increased until the third week of vaccination and then decreased slightly from the fourth week. However, it was confirmed that all of the examples showed high antibody titer during the experiment period, showing high defense (see Fig. 8).

The antibody titers to IgG (see FIG. 9A), IgG1 (see FIG. 9B), and IgG2a (see FIG. 9C) were measured using LPS as an antigen. As a result, IgG1 of Example 1 High antibody titers were observed in all groups except for the antibody titer during the experiment. In the case of vaccination, the immunized group of Example 2 against IgG and IgG1 showed significantly higher antibody titer than that of the immunized group of Example 1, showing the same tendency when whole cell antigen was used. In addition, the antibody level of Example 2 gradually increased during the experimental period from the first week of vaccination, while the antibody level of Example 1 increased until the third week of vaccination and then decreased slightly from the fourth week of vaccination. As shown in Fig. However, at the 4th week, the antibodies of the examples tended to show a difference in the case of LPS in comparison of the antigens. This suggests that Example 1, in which O-polysaccharide and core oligosaccharide were completely removed, showed relatively lower antibody titer when LPS was used as an antigen.

Statistical analysis

One-way ANOVA was used for statistical analysis to see the significant difference between the experimental groups. p <0.05 was considered significant, and multiple comparisons of Bonferroni were performed to compare differences between groups.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Biotechnology Research Institute KCTC12797BP 20150421 Korea Biotechnology Research Institute KCTC12798BP 20150421

<110> Industry Academic Cooperation Foundation of Kangwon National University <120> SALMONELLA ENTERITIDIS MUTANT STRAINS HID2092 AND HID2114 AND          PHARMACEUTICAL COMPOSITION USING THE SAME <130> pn1503-075 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 489 <212> DNA <213> rfaH gene sequence <400> 1 atgcaatcct ggtatttact gtactgcaaa cgcgggcaac ttcagcgtgc tcaggaacac 60 ctcgaaagac aagcggtaag ttgcctgaca ccgatgatca ccctggaaaa aatggtacgc 120 ggaaaacgta cctccgtcag cgaaccgctc tttcctaatt atctgttcgt tgaatttgat 180 ccggaagtga tacataccac tacaatcaac gccacgcgcg gcgtcagcca ttttgtgcgc 240 tttggcgcgc atcctgcgat cgtgccttcc agcgttattc atcagctttc tatctacaag 300 cccgaaggcg ttgtcgatcc tgaaaccccc tatcccggcg atagcgtcat catcacggaa 360 ggcgcatttg aagggctgaa agcgattttt accgaaccgg atggcgaaac gcgttcgatg 420 ttactgctta atttactcaa taaagaagtg aagcagagcg taaaaaacac cggttttcgc 480 aagatttag 489 <210> 2 <211> 990 <212> DNA <213> msbB gene sequence <400> 2 agcagaccct ggaaaagcat ggaaaccaaa aaaaataata gtgagtatat ccctgaattc 60 gaaaaatcct ttcgctatcc gcagtattgg ggcgcctggt tgggcgcggc ggcaatggcg 120 gggattgcat taacaccggc atcattccgt gaccctttgc tggcgacgct ggggcgcttt 180 gccggacggc tggggaagag ttctcgtcgc cgggcgctaa ttaatctgtc tttgtgcttt 240 ccgcagcgta gcgaagctga gcgcgaagcg attgtcgatg agatgttcgc caccgcgcca 300 caggcaatgg cgatgatggc tgagttggcg atgcgcggtc cgaaaaaaat tcaacagcgt 360 gttgactggg aaggtctgga aatcattgag gagatgcgtc gtaacgacga aaaagtcatt 420 tttctcgtac cgcatggctg gggcgtcgac attccagcta tgctgatggc ctctcagggg 480 caaaaaatgg cggcgatgtt tcataatcag ggtaatccgg tttttgacta tatctggaac 540 accgtgcgtc ggcgttttgg cggacgtttg catgcgcgca atgacgggat taaacccttt 600 attcagtctg ttcgtcaggg ctactggggt tactacctgc cggaccagga tcatggcccg 660 gagcatagtg aattcgttga tttctttgcg acatacaaag cgacgctgcc cgcgattggt 720 cggctgatga aagagtgccg cgcacgcgtg ataccgcttt tcccggtgta taatggtaaa 780 acgcatcgcc tgactatcca gattcgcccg ccaatggacg atctgctcac ggctgacgac 840 cacactatcg ccagacggat gaacgaagag gtcgaaattt ttgtcggccc gcatccggaa 900 cagtacacct ggatcctgaa gctgctcaaa acccgcaagc caggcgagat tcagccgtat 960 aagcgtaaag atctttatcc catcaaataa 990

Claims (7)

A pharmaceutical composition for preventing or treating salmonellosis, which comprises as an active ingredient a Salmonella enteritidis mutant strain in which the msbB gene consisting of the nucleotide sequence of SEQ ID NO: 2 is inactivated on the chromosome of Salmonella enteritidis. The method according to claim 1,
Wherein the inactivation of the gene is due to a deletion of a part or all of the gene of interest. delete The method according to claim 1,
Wherein the mutant strain Salmonella enteritidis in which the msbB gene is inactivated is a strain of Accession No. KCTC 12798BP. delete The method according to claim 1,
The pharmaceutical composition for preventing or treating salmonellosis, wherein the composition is a live vaccine composition. A method for preventing or treating salmonellosis by inoculating birds with the mutant Salmonella enteritidis strain of claim 1.

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