WO2022270953A1 - Salmonella typhimurium strain having yjek gene deleted therefrom and salmonella vaccine composition comprising same - Google Patents
Salmonella typhimurium strain having yjek gene deleted therefrom and salmonella vaccine composition comprising same Download PDFInfo
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- WO2022270953A1 WO2022270953A1 PCT/KR2022/008979 KR2022008979W WO2022270953A1 WO 2022270953 A1 WO2022270953 A1 WO 2022270953A1 KR 2022008979 W KR2022008979 W KR 2022008979W WO 2022270953 A1 WO2022270953 A1 WO 2022270953A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0275—Salmonella
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a YjeK gene-deleted Salmonella typhimurium strain and a use thereof, and more particularly to a novel Salmonella typhimurium strain; And vaccine compositions, immunogenic compositions and feed compositions comprising the same; it relates to.
- Salmonella is a gram-negative facultative anaerobic bacterium belonging to Enterobacteriaceae, and is a bacillus that does not form spores. Salmonella is a pathogenic microorganism that infects humans as well as various livestock including pigs, cows, chickens, etc. to cause diseases. It infects humans to cause food poisoning and causes various types of Salmonellosis in animals.
- Salmonella enterica is classified by serological classification as Salmonella Typhi, which causes typhoid fever, Salmonella Typhimurium, which is the causative agent of typhoid fever, Salmonella Enteriditis, which is an enteritis bacterium, and poultry It includes serospecies such as Salmonella Gallinarum, the causative bacterium of typhus, and Salmonella Pullorum, the causative bacterium of Chubaekri. Salmonella includes zoonotic agents that, depending on the serotype, can cause disease not only in humans but also in livestock.
- Salmonella typhimurium and Salmonella enteritidis are zoonotic infectious agents that cause food poisoning in humans and cause salmonellosis in livestock such as cattle, chickens and pigs, resulting in acute or chronic enteritis and sepsis, further pneumonia, It causes arthritis, miscarriage, etc.
- Salmonella galinarum and Salmonella florum are poultry-specific infections that can cause poultry typhus and chubaekri, which can cause mass mortality in poultry. Salmonella florum has no clinical symptoms even when infected in the sex system, but is transmitted from egg to hatched chicks, causing chubaekri, and the highest mortality rate occurs at 1-2 weeks of age.
- antibiotics are mainly used to prevent and treat Salmonella infection, but Salmonella often penetrates into cells when animals are infected and proliferates, making it difficult for antibiotics, drugs, and other probiotics to penetrate and act.
- the problem of antibiotic susceptibility has been occurring, and the cause of this has been identified as the misuse of antibiotics, and antibiotics have not been used sufficiently.
- antibiotics used as growth promoters in compound feeds for industrial animals began to be banned, and antibiotic use began to be regulated in the European Union from 2006 and in Korea from the second half of 2011.
- the use of antibiotics is gradually decreasing.
- the use of antibiotics to control bacteria is reduced, the occurrence of bacterial diseases is increasing, so there is an increasing demand for methods of controlling bacterial diseases without using antibiotics.
- the present inventors completed the present invention by developing a Salmonella typhimurium strain in which the YjeK gene was deleted and confirming its remarkable Salmonella infection preventive effect.
- an object of the present invention is to provide a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide sequence of SEQ ID NO: 1 is deleted.
- Another object of the present invention is a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a group consisting of a concentrate of the culture and a dry product of the culture To provide a composition comprising at least one selected from.
- Another object of the present invention is a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dried product of the culture
- a method for preventing or treating salmonellosis comprising; administering at least one selected from the group to a subject in need thereof.
- the present invention provides a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide sequence of SEQ ID NO: 1 is deleted.
- the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of Provided is a vaccine composition for preventing or treating Salmonellosis, including more than one species.
- the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of An immunogenic composition against Salmonella comprising more than one species is provided.
- the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of It provides a feed composition for preventing or improving salmonellosis comprising more than one species.
- the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of It provides a feed additive composition for preventing or improving salmonellosis comprising more than one species.
- the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of It provides a method for preventing or treating salmonellosis, including the step of administering at least one species to a subject in need thereof.
- the YjeK gene-deleted Salmonella typhimurium strain according to the present invention can effectively prevent Salmonella infection and significantly increase the survival rate of individuals. This means that diseases related to Salmonella infection can be effectively prevented when the YjeK gene-deleted Salmonella typhimurium strain of the present invention is used as a vaccine. It can be used in various fields of control.
- Figure 1A is a diagram showing the results of analyzing the growth of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- Figure 1B is a diagram showing the results of analyzing the motility of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- Figure 1C is a diagram showing the results of lipopolysaccharide and outer membrane protein profile analysis of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- Figure 1D is a diagram showing the antibiotic susceptibility of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- Figure 2A is a diagram showing the results of analyzing the cell invasion and macrophage viability of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- Figure 2B is a diagram showing the results of analyzing the expression of the gene associated with SPI-1 and SPI-2 T3SS of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- Figure 3A is a diagram showing the results of measuring the spleen weight of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- Figure 3B is a diagram showing the results of measuring the number of bacteria in the spleen of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- Figure 3C is a diagram showing the results of measuring the body weight of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- Figure 4A is a diagram showing the results of measuring serum antibody IgG levels of mice infected with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- Figure 4B is a diagram showing the results of measuring the level of serum antibody IgM in mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- 4C is a diagram showing the results of measuring the IgG2a/IgG1 ratio of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- 5A is a diagram showing the results of measuring the level of the cytokine IFN- ⁇ in splenocytes of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- 5B is a diagram showing the results of measuring the level of the cytokine IL-6 in splenocytes of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- 5C is a diagram showing the results of measuring the level of the cytokine TNF- ⁇ in splenocytes of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- 6A is a diagram showing an immunization test schedule of mice immunized with a ⁇ yjeK mutant strain and a wild-type strain according to the present invention.
- Figure 6B is a diagram showing the results of measuring the body weight of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- Figure 6C is a diagram showing the results of analyzing the liver weight and the number of bacteria in the liver of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- 6D is a diagram showing the results of analyzing the spleen weight and the number of bacteria in the spleen of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- 6E is a diagram showing the results of survival analysis of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
- the present invention provides a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide sequence of SEQ ID NO: 1 is deleted.
- the YjeK gene-deleted Salmonella typhimurium strain of the present invention is represented by the nucleotide sequence of SEQ ID NO: 1 in the yjeK gene of Salmonella typhimurium ST1120 (Kim et al., 2017) isolated in Korea through the ⁇ red recombination method. nucleotides are deleted.
- a variant of the YjeK gene represented by the nucleotide sequence of SEQ ID NO: 1 is included within the scope of the present invention.
- the gene has a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: It means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by 1.
- the "percentage of sequence homology" for polynucleotides is determined by comparing two optimally aligned sequences with a comparison region, wherein 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. may include additions or deletions (i.e., gaps) compared to (not including).
- the strain preferably blocks biochemical activities related to arginine dihydrolase and lysine decarboxylase, but the scope of the present invention is not limited thereto. .
- the strain is AMC (amoxicillin / clavulanic acid), ampicillin (ampicillin, AM), cephalothin (cf), gentamicin (GM), kanamycin (kanamycin, K), neo It is preferable to exhibit antibiotic sensitivity to at least one antibiotic selected from the group consisting of neomycin (N) and ampicillin/sulbactam (SAM).
- the strain preferably has a lower expression of flagellin FliC than the wild-type strain and an increased expression of porin OmpD than the wild-type strain.
- the strain does not induce splenomegaly in the host.
- spleen enlargement means that the volume or weight of the spleen is increased beyond the normal range. Since the spleen is the largest of the lymphoid tissue in the body, when there is a systemic disease of the hematopoietic organ, it reacts with other hematopoietic organs in the body and expands. Splenomegaly is mainly seen in (1) infections (malaria, subacute endocarditis, salmonellosis, etc.), (2) blood diseases (leukemia, anemia, etc.), and (3) metabolic abnormalities.
- the culture means that the YjeK gene-deleted Salmonella typhimurium strain is cultured in a culture medium or culture medium, and the culture is a culture containing the strain.
- the formulation of the culture or culture concentrate is not limited, and for example, the formulation may be liquid or solid.
- the medium contains nutrients required by the microorganism to be cultured, that is, the microorganism to be cultured, and may be a mixture in which a substance for a special purpose is additionally added.
- the medium may also be referred to as an incubator or a culture medium, and is a concept that includes all of a natural medium, a synthetic medium, or a selective medium. It may be obtained by separating from the cultured medium, and the medium may be used without limitation as long as the YjeK gene-deleted Salmonella typhimurium strain can grow.
- the medium is, for example, normal agar medium (or nutrient agar medium; Nutrient agar), TSA (tryptic soy agar) medium, standard agar medium (Standard Methods Agar; Plate Count Agar), lactose medium (lactose broth), BGlB medium ( Brilliant Green lactose Bile Broth), double strength BGlB medium, Endo agar medium (Endo agar), EMB agar medium (Eosin methylene blue agar), normal medium (or nutrient medium; Nutrient Broth), desoxycholate lactose agar medium (Desoxycholate medium) lactose Agar), LB medium (Luria-bertani Broth) or EC medium (EC Broth).
- normal agar medium or nutrient agar medium; Nutrient agar
- TSA tryptic soy agar
- standard agar medium Standard agar medium (Standard Methods Agar;
- the composition may be formulated and used as a wettable powder, granule or capsule, but is not limited to the above type.
- the hydrating agent of the present invention may be prepared by drying and pulverizing the solid medium inoculated with the strain, and then adding and mixing a surfactant, an extender or a nutrient.
- a surfactant polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene alkyl
- the extenders and nutrients include One or two or more selected from the group consisting of soy flour, rice, wheat, ocher, diatomaceous earth, dextrin, glucose,
- the granules of the present invention may use the same surfactants, extenders and nutrients as described above after drying and pulverizing the solid medium inoculated with the strain.
- the granule of the present invention is one or two or more selected from the group consisting of a surface active agent of the strain, an inert carrier, a preservative, a wetting agent, a supply promoter, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protective agent, a buffer and a flow agent It can be prepared by adding more.
- prevention of the present invention means any action that suppresses or delays the onset of a disease by administering a composition.
- treatment of the present invention refers to all activities that improve the symptoms of the disease or inhibit or alleviate the disease and change beneficially by the administration of the composition.
- the composition is intraperitoneal administration, intravenous administration, intramuscular administration, intranasal administration, intra-articular administration, intra-synovial administration, intrathecal administration, intrahepatic administration (intrahepatic) administration, intralesional administration, or intracranial administration is preferable, and more preferably, it may be intraperitoneal administration.
- the vaccine is a veterinary vaccine containing an antigenic material, which is specific for Salmonella and is administered for the purpose of inducing active or passive immunity.
- an antigenic material which is specific for Salmonella and is administered for the purpose of inducing active or passive immunity.
- the vaccine composition according to the present invention can be administered in an immunologically effective amount.
- the "immunologically effective amount” means an amount sufficient to exhibit a preventive effect against a disease associated with a YjeK gene-deleted strain of Salmonella typhimurium and an amount sufficient to not cause side effects or serious or excessive immune reactions, and is administered accurately.
- the concentration varies depending on the specific immunogen to be administered, and can be easily determined by a person skilled in the art according to factors well known in the medical field, such as the age, weight, health, sex of the subject to be vaccinated, sensitivity to drugs of the individual, administration route, and administration method. It can be administered once or several times.
- the vaccine composition according to the present invention may include, in addition to the YjeK gene-deleted Salmonella typhimurium strain as an active ingredient, one or more immune enhancers or excipients or carriers suitable for constituting the vaccine composition.
- An adjuvant that may be included in the vaccine composition of the present invention refers to a substance that enhances the immune response of an injected animal, and many different adjuvants are known to those skilled in the art.
- the immune enhancers include Freund's complete and incomplete immune enhancers, vitamin E, nonionic blocking polymers, muramyl dipeptide, Quil A, mineral oil and non-mineral oil and Carbopol, water-in-oil emulsion immune enhancers, etc., but are limited thereto it is not going to be
- Carriers that may be included in the vaccine composition of the present invention are known to those skilled in the art, and include, but are not limited to, proteins, sugars, and the like.
- the above carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions.
- non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate.
- Aqueous carriers include water, alcoholic/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.
- Carriers for intravenous injection include electrolyte replenishers, liquid and nutritional supplements, and the like, such as those based on Ringer's dextrose.
- the vaccine composition of the present invention may further contain preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like.
- the preservatives include formalin, thimerosal, neomycin, polymyxin B and amphotericin B, and the like.
- the vaccine composition of the present invention may include one or more suitable emulsifiers, such as Span or Tween.
- the vaccine composition of the present invention may include a protecting agent, and a protecting agent known in the art may be used without limitation, which may include lactose (LPGG) or trehalose (TPGG), It is not limited thereto.
- a feed composition for preventing or improving salmonellosis comprising at least one member selected from the group consisting of; Or a feed additive composition for preventing or improving salmonellosis; it may be.
- feed means any natural or artificial composition, one meal, etc., or a component of the one meal meal eaten by animals, and includes the YjeK gene-deleted Salmonella typhimurium strain according to the present invention as an active ingredient.
- the feed may be prepared with various types of feed known in the art, and preferably may include concentrated feed, roughage and / or special feed, but is not limited thereto.
- the feed additive is aimed at various effects such as alleviation of disease symptoms in animals, nutrient supplementation and weight loss prevention, improvement of digestibility of fiber in feed, oil quality improvement, reproduction disorder prevention and conception rate improvement, and summer high temperature stress prevention.
- the feed composition and feed additive composition of the present invention correspond to supplementary feed under the Feed Control Act, and include mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, and trace minerals such as zinc, copper, cobalt, and selenium.
- Mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, and trace minerals such as zinc, copper, cobalt, and selenium.
- Mineral preparations such as kerotene, vitamins A, D, E, nicotinic acid, and vitamin B complex
- protective amino acids such as methionine and lysine
- protective fatty acids such as calcium salts of fatty acids
- probiotics lactic acid bacteria
- yeast cultures lactic acid bacteria
- molds Live bacteria such as fermentation products, yeast agents, and the like may be further included.
- the enriched feed includes seed fruits including grains such as wheat, oats and corn, bran including rice bran, bran, barley bran, etc. as a by-product obtained by refining grains, soybeans, fluids, sesame seeds, linseed, coco Fish meal, which is a by-product obtained from oil extraction of palms, residual starch, which is the main component of starch residue, which is the remainder after removing starch from sweet potatoes, potatoes, etc., fish meal, fish residue, and fresh liquid obtained from fish Fish soluble, meat meal, blood meal, feather meal, skim milk powder, animal feed such as dry whey, yeast, chlorella, seaweed but not limited thereto.
- seed fruits including grains such as wheat, oats and corn, bran including rice bran, bran, barley bran, etc.
- bran including rice bran, bran, barley bran, etc.
- by-product obtained by refining grains soybeans, fluids, sesame seeds, linseed, coco Fish meal
- Forage among the feed includes grass feed such as wild grass, grass, green cutting, turnip for feed, beet for feed, root vegetables such as Lutherbearer, a type of turnip, raw grass, green crops, grains, etc. are filled in a silo and fermented with lactic acid It includes, but is not limited to, silage, which is a stored feed, grass, hay made by cutting down grass, straw of breeding crops, and leaves of leguminous plants.
- Special feeds include mineral feeds such as oyster shells and rock salt, urea feeds such as urea or its derivative, diureide isobutane, and supplements for ingredients that are likely to be insufficient when only natural feed ingredients are mixed, or formulated feeds to improve the storability of feeds.
- feed additives and dietary supplements which are substances added in small amounts, but are not limited thereto.
- a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dried form of the culture It provides a method for preventing or treating salmonellosis comprising; administering at least one selected from the group consisting of to a subject in need thereof.
- the YjeK gene-deleted Salmonella typhimurium strain is intravenous administration, intramuscular administration, intranasal administration, intra-articular administration, intra-synovial administration, water Intrathecal administration, intrahepatic administration, intralesional administration or intracranial administration is preferred, and intramuscular or intranasal administration is more preferred, but is not limited thereto.
- the method for preventing or treating salmonellosis of the present invention it is preferable to increase the immune response in vivo by administering a Salmonella typhimurium strain in which the YjeK gene is deleted, and more specifically, the antibody regulates the expression of factors related to the immune response. It is desirable to do
- Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.
- ST1120 wild-type Salmonella enterica serovar Typhimurium 1120 isolated in Korea was used.
- S. Typhimurium 14028 strain ST2173 transformed with the plasmid pBBR1-MCS4 was used.
- the ST2173 strain contains an ampicillin resistance gene.
- Bacterial strains were cultured in Luria-Bertani (LB) broth (Duchefa, Haarlem, The Netherlands) at 37°C with 150 ⁇ g/ml ampicillin (Duchefa) when necessary unless otherwise specified. All antibiotics were purchased from Sigma-Aldrich (St. Louis, MO, USA). The biochemical properties of the bacteria were confirmed using the API 20E kit (bioMerieux, Inc., Durham, NC, USA).
- a ⁇ yjeK mutant strain in which the yjeK gene was deleted was generated through site-directed mutagenesis using the ⁇ red recombination method using plasmids pTP233, pKD3 and pCP20. Specifically, ST1120 containing pTP233 was transformed with a chloramphenicol resistance gene cassette flanked by sequences homologous to the yjeK gene to prepare ST2160 strain. Homologous recombination between the chromosome yjeK and the CM resistance cassette in strain ST2160 was confirmed by diagnostic polymerase chain reaction after plating on LB agar containing 40 ⁇ g/mL chloramphenicol.
- a chloramphenicol-sensitive strain was selected, and the chloramphenicol-resistance gene was removed from the chromosome to obtain strain ST2161.
- the pCP20 plasmid was removed by culturing the chloramphenicol-sensitive strain at 42° C., and deletion of the yjeK and CM cassettes was further verified using diagnostic PCR.
- Bacterial antibiotic susceptibility was assessed using the disk diffusion test on Mueller-Hinton agar (Becton Dickinson, Sparks, MD, USA). Bacterial cells were incubated in LB broth at 37°C for 16-18 hours. The optical density (OD 600 ) at 600 nm of the cultured cells was 0.6-0.7, and the number of cultured cells corresponded to 5 ⁇ 10 7 cells. Colony forming units (CFU)/mL were smeared on Mueller-Hinton (MH) agar using sterile swabs prior to placement of antibiotic discs (BBLTM Sensi-DiscTM, Becton Dickinson).
- MH Mueller-Hinton
- antibiotics AMC amoxicillin/clavulanic acid
- 10 ⁇ g of ampicillin (AM) ampicillin
- CF cephalothin
- 10 ⁇ g of gentamicin (GM) 10 ⁇ g of gentamicin (GM), kanamycin, K
- AMC ampicillin/clavulanic acid
- CF cephalothin
- GM gentamicin
- K Antibiotic susceptibility to 30 ⁇ g, nalidixic acid (NA) 30 ⁇ g, neomycin (N) 30 ⁇ g
- SAM ampicillin/sulbactam
- SXT sulfamethoxazole/trimethoprim
- lysing solution 0.1 M sodium dodecyl sulfate, 50 mM Tris base, 0.128 M NaOH
- P/C/I solution phenol:chloroform:isoa
- the LPS fraction was precipitated overnight at -20 °C and centrifuged at 10,000 xg. LPS precipitated for 20 minutes was resuspended in sterile water and treated with DNase (Sigma-Aldrich) and RNase (Promega, Madison, WI, USA) at 37°C for 1 hour.
- DNase Sigma-Aldrich
- RNase Promega, Madison, WI, USA
- the LPS solution was mixed with the P/C/I solution and centrifuged at 10,000 xg for 20 minutes.
- Purified LPS was suspended in sterile water and quantified using the Pierce LAL Chromogenic Endotoxin Quantification Kit (Thermo Scientific Inc., IL, USA). A 5 ⁇ g LPS sample was loaded on a 12% DOC-PAGE (deoxycholate-polyacrylamide gel electrophoresis) gel and the LPS profile was visualized.
- Bacterial OMPs were isolated and analyzed. Specifically, bacterial cells cultured in LB broth were harvested by centrifugation at 2,500 xg for 20 minutes. Bacterial cell pellets were suspended in 10 mM N-2-hydroxyethylpiperazine N′-2-ethanesulfonic acid and sonicated with a Vibra-cell ultrasonic liquid processor (Sonics & Materials Inc., Newtown, CT, USA). The bacterial membrane fraction of cell lysates was collected by ultracentrifugation using a 45 Ti rotor (Beckman Coulter, Brea, CA, USA) at 100,000 xg.
- the membrane fraction was resuspended in a buffer containing 1% N-lauroylsarcosine solution (Sarkosyl, Sigma-Aldrich) to solubilize and remove inner membrane proteins.
- the sarcosyl-treated OMP fraction was purified using ultracentrifugation as described above and finally resuspended in phosphate buffered saline.
- the concentration of OMP was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific Inc.). 10 ⁇ g of OMP was resolved using 12% SDS-PAGE and visualized by staining with Coomassie Brilliant Blue.
- Protein bands of interest were analyzed by peptide mass fingerprinting method. Briefly, proteins were extracted from SDS-PAGE gels and then digested with trypsin (Promega). digested peptide; and ⁇ -cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid (Sigma-Aldrich), and the peptide mixture was subjected to matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Microflex LRF 20, Bruker Daltonics , MA, USA).
- Mass spectra were collected in the m/z range of 600-3,000 and internally calibrated by the trypsin autodigestion peak (m/z 842.510, 2211.1046). Mass spectra were analyzed using the MASCOT server version 2.3 (Matrix Science, London, UK). Proteins with a MASCOT score >73 were considered significantly matched to the target protein (P ⁇ 0.05).
- RNA protect Bacteria Reagent (Qiagen, Hilden, Germany) or RNAlater stabilizing solution (Ambion, Austin, TX, USA) according to the manufacturer's instructions. processed. Bacterial total RNA was isolated using the RNeasy mini kit (Qiagen) and residual chromosomal DNA was removed using the TURBO DNA-free kit (Ambion). RNA was reverse transcribed into cDNA using RNA to cDNA EcoDry Premix with random hexamers (Clontech Laboratory, CA, USA), and cDNA corresponding to 10 ng of input RNA was used as a template in each qPCR. Primers were designed using Primer Express Software version 3.0 (Applied Biosystems, MA, USA), and the designed primer sequences are shown in Table 2 above.
- qPCR was performed using a StepOnePlus real-time PCR instrument (Applied Biosystems) with SYBR Green reagent (Power SYBR Green PCR Master Mix, Applied Biosystems) to detect amplified PCR products.
- SYBR Green reagent Power SYBR Green PCR Master Mix, Applied Biosystems
- the relative expression level of each gene was normalized to that of gyrB and expressed as the average of three tests using independently extracted RNA samples.
- HeLa human epithelial cells and RAW264.7 murine macrophages were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS (fetal bovine serum), at densities of 1 ⁇ 10 5 and 2 ⁇ 10 5 cells/well. were inoculated into 24-well culture plates, respectively.
- Salmonella cells cultured in LB broth overnight were added to animal cells at a multiplicity of infection of 100. After 30 min of bacterial infection, cells were washed 3 times with PBS and supplemented with fresh DMEM containing gentamicin (100 ⁇ g/mL) for 1.5 h. The medium was replaced with fresh DMEM containing 10 ⁇ g/mL gentamicin for the remainder of the infection period to remove extracellular bacteria.
- DMEM Dulbecco's Modified Eagle's Medium
- FBS fetal bovine serum
- infected HeLa cells were lysed using 1% Triton X-100 2 hours post infection, cell lysates were diluted and plated on LB agar.
- RAW264.7 cells infected in the survival assay were lysed 10 hours after infection, and intracellular bacteria were counted in the same way as described for the invasion assay.
- LD 50 half-maximal lethal dose
- In vivo toxicity testing was performed using groups of 4 BALB/c mice infected intraperitoneally with bacterial cells at 10 4 CFU/dose. Mice were sacrificed on day 14 post infection (dpi) and body and spleen weights were measured. Spleens were subsequently homogenized using a TissueLyser II (Qiagen, USA) at 30 Hz for 1 minute, and lysates were serially diluted. To enumerate bacterial cells, serial dilutions were plated on Salmonella-Shigella (SS) agar (Difco, Becton Dickinson, MD, USA).
- SS Salmonella-Shigella
- mice Female BALB/c mice were divided into groups of 6 mice each and immunized with wild-type and ⁇ yjeK mutant strains by intraperitoneal injection with 10 3 CFU/dose and 10 4 CFU/dose, respectively.
- Negative control (N control) was inoculated with PBS instead of the strain. Blood was collected at 0, 7, 14, 28 and 35 days after mouse immunization and serum was stored at -20 °C until use. After 28 days, mice were orally administered with 10 8 CFU/dose (protection assay) or 10 10 CFU/dose (survival assay) of the ST2173 strain.
- a positive control was challenged with the same dose of ST2173 after administration of PBS.
- mice In the mouse protection assay, antigen-challenged mice were sacrificed 35 days after immunization. Organs including liver and spleen were collected from sacrificed mice, and plated on SS agar. In mouse survival assays, challenged mice were monitored for an additional 24 days and mortality was recorded.
- IgG and IgM antibody responses in serum were measured using ELISA as previously described. Optimal concentrations of serum and enzyme conjugate were determined by a checkerboard test using triplicate sera from immunized and non-immunized mice. S. by selecting the best binding ratio between serum and enzyme conjugates. Typhimurium-specific IgG, IgM and IgG subclass (IgG1 and IgG2a) antibody responses were measured. wild type S. OMP prepared from Typhimurium was added to a microplate (Thermo Scientific Inc.) at 0.5 ⁇ g/well and incubated overnight at 4°C. Serum from immunized mice was diluted 1:200 and added to the coated wells.
- microplate was washed with PBS (TBST) containing Tween 20, and horseradish peroxidase-labeled goat anti-mouse IgG (1:30,000 dilution; Bethyl Laboratories, TX, USA), anti-mouse IgM (1 :35,000 dilution, Bethyl Laboratories), anti-mouse IgG1 (1:25,000 dilution, Bethyl Laboratories) or anti-mouse IgG2a (1:30,000 dilution, Bethyl Laboratories) secondary antibody was treated. Unbound secondary antibody was washed 3 times with 0.05% PBST.
- spleen cells were isolated from the spleen and counted. 2X10 5 cells/well were plated in complete medium consisting of RPMI-1640 (GenDEPOT, TX, USA), 10 mM HEPES (Duchefa) and 10% FBS. The plated cells were killed by LPS (5 ⁇ g/mL, Sigma-Aldrich) or heat. Typhimurium cells (HKC; 10 8 CFU/0.1 mL) were treated and cultured for 48 hours at 37°C and 5% CO 2 conditions. The supernatant of the cultured cells was collected and cytokines were measured. The supernatant was stored at -20 °C until cytokine measurement.
- IFN- ⁇ , IL-6 and TNF- ⁇ were determined using a cytokine ELISA kit (MAXTM Standard, BioLegend, Inc., CA, USA) according to the manufacturer's instructions. Cytokine concentrations were calculated using Four Parameter Logistic Curve software (MyAssays Ltd., https://www.myassays.com/).
- the yjek gene of the wild-type strain ST1120 is represented by the nucleotide sequence of SEQ ID NO: 2, and the nucleotide sequence of SEQ ID NO: 1 was deleted from the wild-type yjek gene using the ⁇ red recombination method. That is, the ⁇ yjeK mutant strain includes the yjek gene in which the nucleotide of SEQ ID NO: 1 is deleted. Since the efp gene is located near the deletion site, the expression of efp was compared between the wild-type and deletion strains. Transcription of efp was hardly affected by the deletion in the 1yjeK mutant strain, ruling out the possibility of a polar effect on EF-P activity in the ⁇ yjeK mutant strain.
- FIGS. 1A and B The results of analyzing the growth and motility of the ⁇ yjeK mutant strain are shown in FIGS. 1A and B, respectively.
- the ⁇ yjeK mutant strain blocked the biochemical activities related to arginine dihydrolase and lysine decarboxylase, thereby reducing arginine and lysine decapsulants in the mutant strain. It was confirmed that it represents an altered metabolic pathway of reboxylation.
- the OmpA induces strong immunogenicity in Salmonella infection, and less porin OmpD is produced, reducing bacterial permeability to oxidizing molecules and promoting bacterial survival in the host.
- Bacterial OMPs that form porins and efflux pumps affect bacterial resistance to antibiotics by reducing permeability (porins) or increasing drug export (efflux pumps). Therefore, as an increase in porin OmpD was observed in the ⁇ yjeK mutant strain, resistance to antibiotics was further confirmed. The results of confirming resistance to antibiotics are shown in FIG. 1D.
- the ⁇ yjeK mutant strain was susceptible to other antibiotics except NA, SXT and TE.
- the inhibitory regions of GM, N, K, and AMC were up to 77% (21.0 ⁇ 0.0 mm), 45% (18.9 ⁇ 0.5 mm), 36% (21.8 ⁇ 1.1 mm), and 34%, respectively. % (27.5 ⁇ 0.7 mm).
- the ⁇ yjeK mutant strain entered host epithelial cells 3.73 times faster than wild-type bacteria at 2 hours after infection, but failed to fully proliferate in macrophages 10 hours after infection.
- mice infected with the ⁇ yjeK mutant strain After sacrifice of mice infected with the ⁇ yjeK mutant strain, spleen and body weights were measured. The number of bacteria in the isolated spleen was also counted. The results of confirming the weight of the mouse spleen and the number of bacteria in the spleen are shown in FIGS. 3A and B, respectively. In addition, the result of measuring the body weight of the sacrificed mouse is shown in Figure 3C.
- mice infected with the ⁇ yjeK mutant strain were also clearly different from those of the wild-type infected group.
- the weight of the spleen was significantly reduced ( ⁇ yjeK, 0.34 ⁇ 0.05 g vs. wild-type, 0.72 ⁇ 0.12 g), whereas splenomegaly caused by the Salmonella strain was observed in the infection with the wild-type strain. .
- the number of bacteria in the spleen of the ⁇ yjeK mutant strain treatment group was 4.6X10 4 CFU/g, which was significantly lower than that of the wild-type strain treatment group (2.6X10 5 CFU/g).
- the immunological effect of the ⁇ yjeK mutant was measured by the level of serum antibodies (IgG and IgM) produced after infection in BALB/c mice.
- the results of analyzing the expression levels of IgG and IgM are shown in FIGS. 4A and B, respectively.
- the levels of IgG and IgM antibodies were similar for groups infected with wild-type and ⁇ yjeK mutant strains up to 14 dpi. At 28 dpi, immunization with the ⁇ yjeK mutant strain resulted in a 1.7- and 1.5-fold increase in the levels of IgG and IgM antibodies compared to 14 dpi, respectively.
- the IgG2a/IgG1 ratio is commonly used as a surrogate marker for the immune balance between T helper (Th)1 and Th2 responses to microbial infections.
- the IgG2a/IgG1 ratio of the immunized mice was measured, and the results are shown in FIG. 4C.
- the IgG2a/IgG1 ratio of the mice immunized with the ⁇ yjeK mutant strain ranged from 2.32 (28 dpi) to 3.66 (14 dpi), and the IgG2a/IgG1 ratio of the group infected with the wild-type strain was 5.16 (28 dpi). ) to 5.65 (14 dpi).
- the spleen of the mouse was collected at 28 dpi, and the splenocytes isolated from the spleen were treated with S. Typhimurium LPS and HKC to estimate the cytokine production level for the Salmonella immunogen.
- S. Typhimurium LPS and HKC The results of analyzing the cytokines IFN- ⁇ , IL-6 and TNF- ⁇ in the immunized mouse splenocytes are shown in FIGS. 5A to C, respectively.
- the cytokines IFN- ⁇ , IL-6 and TNF- ⁇ were all increased in response to LPS or HKC, regardless of the bacterial strain used for immunization.
- immunization with the ⁇ yjeK mutant strain promoted IFN- ⁇ production to a greater extent for LPS (2.24-fold) and HKC (1.5-fold) than was observed for wild-type Salmonella (Fig. 5A).
- production of the pro-inflammatory cytokines IL-6 and TNF- ⁇ was induced to a lesser extent after immunization with the ⁇ yjeK mutant strain for both LPS and HKC treatment (FIGS. 5B and C).
- mice immunized with the ⁇ yjeK mutant strain induce IgG and IgM antibodies without signs of systemic infection in the host organ, and at the same time modulate cytokine production in response to challenge with immunogenic antigens of Salmonella.
- the preventive effect of immunization with the ⁇ yjeK mutant strain was evaluated in BALB/c mice.
- the ⁇ yjeK mutant strain which is a candidate vaccine, was immunized by intraperitoneal administration to mice.
- Immunized mice 28 dpi
- ST2173 wild-type S. Typhimurium 2173
- ST2173 wild-type S. Typhimurium 2173
- ST2173 Clinical symptoms of mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain were observed. As a result, ST2173-infected mice without prior vaccination showed typical clinical signs of Salmonella infection, including coarse fur, hard, closed eyes, anorexia, hunched posture, tremors, weight loss and death.
- mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain was measured, and the results are shown in FIG. 6B.
- mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain was reduced by about 77% by ST2173 infection.
- mice immunized with the ⁇ yjeK mutant strain showed weight gain for 7 days after infection with ST2173, similar to healthy mice not injected with the Salmonella strain.
- mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain were sacrificed, the weight of the liver and spleen was measured, and the number of bacteria in the liver and spleen was analyzed.
- the results of analyzing the weight and number of bacteria in the liver are shown in FIG. 6C, and the results of analyzing the weight and number of bacteria in the spleen are shown in FIG. 6D.
- mice immunized with the candidate vaccine showed no signs of liver and spleen enlargement, whereas challenged mice without prior vaccination had enlarged livers and spleens.
- mice immunized with the candidate vaccine (3.0 X 10 3 CFU/g liver and 4.8 X 10 3 CFU/g spleen) was higher than that of mice immunized with wild-type Salmonella (2.3 X 10 4 CFU/g liver and 7.5 ⁇ 10 4 CFU/g spleen) and unimmunized mice (positive challenge control: 3.9 X 10 5 CFU/g liver and 2.5 X 10 6 CFU/g spleen).
- the prophylactic effect of the candidate vaccine was evaluated by monitoring the survival of immunized mice after oral infection with ST2173 at a bacterial dose of 100 LD 50 .
- the evaluation results are shown in Fig. 6E.
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Abstract
The present invention relates to a Salmonella typhimurium strain having YjeK gene deleted therefrom and to uses thereof. More specifically, the present invention relates to: a novel Salmonella typhimurium strain; and a vaccine composition, an immunogenic composition, and a feed composition comprising same. It was confirmed that the Salmonella typhimurium strain having YjeK gene deleted therefrom according to the present invention can effectively prevent Salmonella infection, thereby remarkably increasing a survival rate of an individual. This means that when the Salmonella typhimurium strain having YjeK gene deleted therefrom of the present invention is used as a vaccine, diseases related to Salmonella infection can be effectively prevented such that the present invention can be used in various ways in the field of livestock disease control.
Description
본 발명은 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주 및 이의 용도에 관한 것으로, 보다 상세하게는 신규 살모넬라 티피뮤리움 균주; 및 이를 포함하는 백신 조성물, 면역원성 조성물 및 사료 조성물;에 관한 것이다.The present invention relates to a YjeK gene-deleted Salmonella typhimurium strain and a use thereof, and more particularly to a novel Salmonella typhimurium strain; And vaccine compositions, immunogenic compositions and feed compositions comprising the same; it relates to.
살모넬라(Salmonella)는 장내세균과에 속하는 그람음성의 통성 혐기성 세균으로, 아포를 형성하지 않는 간균이다. 살모넬라균은 사람뿐만 아니라 돼지, 소, 닭 등을 비롯한 다양한 가축에 감염되어 질병을 유발하는데, 사람에게 감염되어 식중독을 유발하고 동물에게 있어서는 다양한 살모넬라증(Salmonellosis)을 유발하는 병원성 미생물이다. 살모넬라 엔테리카(Salmonella enterica)는 혈청학적 구분에 의해 장티푸스를 일으키는 살모넬라 타이피(Salmonella Typhi), 쥐티푸스의 원인균인 살모넬라 티피뮤리움(Salmonella Typhimurium), 장염균인 살모넬라 엔테리티디스(Salmonella Enteriditis), 가금티푸스의 원인균인 살모넬라 갈리나룸(Salmonella Gallinarum), 추백리 원인균인 살모넬라 플로럼(Salmonella Pullorum) 등의 혈청종을 포함한다. 살모넬라균은 혈청형에 따라 인간뿐만 아니라 가축에게도 질병을 일으킬 수 있는 인수공통 감염원을 포함한다. 살모넬라 티피뮤리움과 살모넬라 엔테리티디스는 인수공통 감염원으로 사람에게 있어서는 식중독의 원인이 되며, 소, 닭, 돼지 등의 가축에 있어서는 살모넬라증(salmonellosis)을 일으켜 급성 또는 만성 장염과 패혈증, 더 나아가 폐렴, 관절염, 유산 등을 발생시킨다. 살모넬라 갈리나룸과 살모넬라 플로럼은 가금 특이적 감염균으로 가금티푸스와 추백리를 일으켜 가금류의 대량 폐사를 유발할 수 있다. 살모넬라 플로럼은 성계에서는 감염되어도 임상 증상이 없으나 부화된 후대 병아리에게 난계대 전염되어 추백리를 일으키며, 생후 1-2주령에서 가장 높은 폐사율이 나타난다. 주로 회백색의 설사 증세를 보이면서 항문을 막아 배분 곤란을 일으켜 복부가 팽창하고, 그로 인한 패혈증으로 폐사한다. 추백리와 같은 경우 상업화된 예방책이 없으며, 감염된 개체의 색출 및 도태를 통해서만 질병의 예방이 가능하다. Salmonella ( Salmonella ) is a gram-negative facultative anaerobic bacterium belonging to Enterobacteriaceae, and is a bacillus that does not form spores. Salmonella is a pathogenic microorganism that infects humans as well as various livestock including pigs, cows, chickens, etc. to cause diseases. It infects humans to cause food poisoning and causes various types of Salmonellosis in animals. Salmonella enterica is classified by serological classification as Salmonella Typhi, which causes typhoid fever, Salmonella Typhimurium, which is the causative agent of typhoid fever, Salmonella Enteriditis, which is an enteritis bacterium, and poultry It includes serospecies such as Salmonella Gallinarum, the causative bacterium of typhus, and Salmonella Pullorum, the causative bacterium of Chubaekri. Salmonella includes zoonotic agents that, depending on the serotype, can cause disease not only in humans but also in livestock. Salmonella typhimurium and Salmonella enteritidis are zoonotic infectious agents that cause food poisoning in humans and cause salmonellosis in livestock such as cattle, chickens and pigs, resulting in acute or chronic enteritis and sepsis, further pneumonia, It causes arthritis, miscarriage, etc. Salmonella galinarum and Salmonella florum are poultry-specific infections that can cause poultry typhus and chubaekri, which can cause mass mortality in poultry. Salmonella florum has no clinical symptoms even when infected in the sex system, but is transmitted from egg to hatched chicks, causing chubaekri, and the highest mortality rate occurs at 1-2 weeks of age. It mainly shows symptoms of grayish-white diarrhea, blocks the anus, causes difficulty in distribution, expands the abdomen, and dies from sepsis. In the case of Chubaekri, there is no commercialized preventive measure, and the disease can be prevented only through the search and culling of infected individuals.
현재 살모넬라 감염증을 예방 및 치료하기 위해 항생제를 주로 사용하고 있으나 살모넬라는 동물에 감염 시 세포내에 침투하여 증식하고 감염하는 경우가 많아 항생제나 약제, 기타 생균제가 침투하여 작용하기는 어려우며, 최근 많은 분야에서 항생제 감수성에 대한 문제가 발생되고 있어 이에 대한 원인을 항생제 오남용으로 규명하고 항생제를 충분히 사용하지 못하게 되었다. 이에 따라 산업동물의 배합 사료 내에 성장촉진제로 사용하는 항생제를 금지하기 시작하였고, 유럽연합에서는 2006년부터, 우리나라에서는 2011년 하반기부터 항생제 사용을 규제하기 시작하였다. 또한 항생제가 없는 안전한 먹거리에 대한 소비자의 요구가 증대되고 있어 점차 항생제 사용이 줄어들고 있다. 세균을 제어하는 항생제의 사용이 줄어듦에 따라 세균성 질병의 발생이 증가하고 있어 항생제를 사용하지 않고 세균성 질병을 제어하는 방법에 대한 요구가 늘어나고 있다.Currently, antibiotics are mainly used to prevent and treat Salmonella infection, but Salmonella often penetrates into cells when animals are infected and proliferates, making it difficult for antibiotics, drugs, and other probiotics to penetrate and act. The problem of antibiotic susceptibility has been occurring, and the cause of this has been identified as the misuse of antibiotics, and antibiotics have not been used sufficiently. Accordingly, antibiotics used as growth promoters in compound feeds for industrial animals began to be banned, and antibiotic use began to be regulated in the European Union from 2006 and in Korea from the second half of 2011. In addition, as consumer demand for antibiotic-free safe food is increasing, the use of antibiotics is gradually decreasing. As the use of antibiotics to control bacteria is reduced, the occurrence of bacterial diseases is increasing, so there is an increasing demand for methods of controlling bacterial diseases without using antibiotics.
이에 본 발명자들은 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주를 개발하고, 이의 현저한 살모넬라균 감염 예방 효과를 확인함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors completed the present invention by developing a Salmonella typhimurium strain in which the YjeK gene was deleted and confirming its remarkable Salmonella infection preventive effect.
따라서 본 발명의 목적은, 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움(Salmonella typhimurium) 균주를 제공하는 것이다.Accordingly, an object of the present invention is to provide a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide sequence of SEQ ID NO: 1 is deleted.
본 발명의 다른 목적은, 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 포함하는 조성물을 제공하는 것이다.Another object of the present invention is a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a group consisting of a concentrate of the culture and a dry product of the culture To provide a composition comprising at least one selected from.
본 발명의 또 다른 목적은, 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 이를 필요로 하는 개체에 투여하는 단계;를 포함하는 살모넬라증의 예방 또는 치료방법을 제공하는 것이다.Another object of the present invention is a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dried product of the culture To provide a method for preventing or treating salmonellosis comprising; administering at least one selected from the group to a subject in need thereof.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주를 제공한다.In order to achieve the above object, the present invention provides a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide sequence of SEQ ID NO: 1 is deleted.
또한 본 발명은 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 포함하는 살모넬라증(Salmonellosis) 예방 또는 치료용 백신 조성물을 제공한다.In addition, the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of Provided is a vaccine composition for preventing or treating Salmonellosis, including more than one species.
또한 본 발명은 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 포함하는 살모넬라균에 대한 면역원성 조성물을 제공한다.In addition, the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of An immunogenic composition against Salmonella comprising more than one species is provided.
또한 본 발명은 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 포함하는 살모넬라증 예방 또는 개선용 사료 조성물을 제공한다.In addition, the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of It provides a feed composition for preventing or improving salmonellosis comprising more than one species.
또한 본 발명은 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 포함하는 살모넬라증 예방 또는 개선용 사료 첨가제 조성물을 제공한다.In addition, the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of It provides a feed additive composition for preventing or improving salmonellosis comprising more than one species.
또한 본 발명은 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 이를 필요로 하는 개체에 투여하는 단계;를 포함하는 살모넬라증의 예방 또는 치료방법을 제공한다.In addition, the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of It provides a method for preventing or treating salmonellosis, including the step of administering at least one species to a subject in need thereof.
본 발명에 따른 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주는 살모넬라균 감염을 효과적으로 예방하여, 개체의 생존율을 현저히 높일 수 있음을 확인하였다. 이는 본 발명의 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주를 백신으로 이용할 경우 살모넬라균 감염 관련 질병을 효과적으로 예방할 수 있음을 의미하는바, 본 발명의 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주는 가축 질병 제어 분야에서 다양하게 활용될 수 있다.It was confirmed that the YjeK gene-deleted Salmonella typhimurium strain according to the present invention can effectively prevent Salmonella infection and significantly increase the survival rate of individuals. This means that diseases related to Salmonella infection can be effectively prevented when the YjeK gene-deleted Salmonella typhimurium strain of the present invention is used as a vaccine. It can be used in various fields of control.
도 1A는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주의 성장을 분석한 결과를 나타낸 도이다.Figure 1A is a diagram showing the results of analyzing the growth of the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 1B는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주의 운동성을 분석한 결과를 나타낸 도이다.Figure 1B is a diagram showing the results of analyzing the motility of the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 1C는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주의 리포폴리사카라이드 및 외막 단백질 프로파일 분석 결과를 나타낸 도이다.Figure 1C is a diagram showing the results of lipopolysaccharide and outer membrane protein profile analysis of the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 1D는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주의 항생제 감수성을 나타낸 도이다.Figure 1D is a diagram showing the antibiotic susceptibility of the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 2A는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주의 세포 침윤 및 대식 세포 내 생존능을 분석한 결과를 나타낸 도이다.Figure 2A is a diagram showing the results of analyzing the cell invasion and macrophage viability of the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 2B는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주의 SPI-1 및 SPI-2 T3SS와 관련된 유잔자의 발현을 분석한 결과를 나타낸 도이다.Figure 2B is a diagram showing the results of analyzing the expression of the gene associated with SPI-1 and SPI-2 T3SS of the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 3A는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 비장 무게를 측정한 결과를 나타낸 도이다.Figure 3A is a diagram showing the results of measuring the spleen weight of mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 3B는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 비장 내 박테리아 수를 측정한 결과를 나타낸 도이다.Figure 3B is a diagram showing the results of measuring the number of bacteria in the spleen of mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 3C는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 체중을 측정한 결과를 나타낸 도이다.Figure 3C is a diagram showing the results of measuring the body weight of mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 4A는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 감염된 마우스의 혈청 항체 IgG의 수준을 측정한 결과를 나타낸 도이다.Figure 4A is a diagram showing the results of measuring serum antibody IgG levels of mice infected with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 4B는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 혈청 항체 IgM의 수준을 측정한 결과를 나타낸 도이다.Figure 4B is a diagram showing the results of measuring the level of serum antibody IgM in mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 4C는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 IgG2a/IgG1 비율을 측정한 결과를 나타낸 도이다.4C is a diagram showing the results of measuring the IgG2a/IgG1 ratio of mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 5A는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 비장세포에서 사이토카인 IFN-γ의 수준을 측정한 결과를 나타낸 도이다.5A is a diagram showing the results of measuring the level of the cytokine IFN-γ in splenocytes of mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 5B는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 비장세포에서 사이토카인 IL-6의 수준을 측정한 결과를 나타낸 도이다.5B is a diagram showing the results of measuring the level of the cytokine IL-6 in splenocytes of mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 5C는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 비장세포에서 사이토카인 TNF-α의 수준을 측정한 결과를 나타낸 도이다.5C is a diagram showing the results of measuring the level of the cytokine TNF-α in splenocytes of mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 6A는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 면역 실험 일정을 나타낸 도이다.6A is a diagram showing an immunization test schedule of mice immunized with a ΔyjeK mutant strain and a wild-type strain according to the present invention.
도 6B는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 체중을 측정한 결과를 나타낸 도이다.Figure 6B is a diagram showing the results of measuring the body weight of mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 6C는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 간 무게 및 간 내 박테리아 수를 분석한 결과를 나타낸 도이다.Figure 6C is a diagram showing the results of analyzing the liver weight and the number of bacteria in the liver of mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 6D는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 비장 무게 및 비장 내 박테리아 수를 분석한 결과를 나타낸 도이다.6D is a diagram showing the results of analyzing the spleen weight and the number of bacteria in the spleen of mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
도 6E는 본 발명에 따른 Δ yjeK 돌연변이 균주 및 야생형 균주로 면역된 마우스의 생존 분석 결과를 나타낸 도이다.6E is a diagram showing the results of survival analysis of mice immunized with the ΔyjeK mutant strain and the wild-type strain according to the present invention.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 양태에 따르면, 본 발명은 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움(Salmonella typhimurium) 균주를 제공한다.According to an aspect of the present invention, the present invention provides a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide sequence of SEQ ID NO: 1 is deleted.
본 발명의 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주는 λ red 재조합 방법을 통해 한국에서 분리된 살모넬라 티피뮤리움 ST1120(Kim et al., 2017)의 yjeK 유전자에서 서열번호 1의 염기서열로 표시되는 뉴클레오티드를 결실시킨 것이다. The YjeK gene-deleted Salmonella typhimurium strain of the present invention is represented by the nucleotide sequence of SEQ ID NO: 1 in the yjeK gene of Salmonella typhimurium ST1120 (Kim et al., 2017) isolated in Korea through the λ red recombination method. nucleotides are deleted.
본 발명의 범위에는 상기 서열번호 1의 염기서열로 표시되는 YjeK 유전자의 변이체가 포함된다. 구체적으로, 상기 유전자는 서열번호 1의 염기서열과 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 것으로, 서열번호 1로 표시되는 염기서열과 실질적으로 동질의 생리활성을 나타내는 서열을 의미한다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.A variant of the YjeK gene represented by the nucleotide sequence of SEQ ID NO: 1 is included within the scope of the present invention. Specifically, the gene has a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: It means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by 1. The "percentage of sequence homology" for polynucleotides is determined by comparing two optimally aligned sequences with a comparison region, wherein 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. may include additions or deletions (i.e., gaps) compared to (not including).
본 발명의 구체예에서, 상기 균주는 아르기닌 디하이드롤라제(arginine dihydrolase) 및 라이신 디카르복실라제(lysine decarboxylation)와 관련된 생화학적 활성을 차단하는 것이 바람직하나, 이에 본 발명의 범위가 제한되지 않는다.In an embodiment of the present invention, the strain preferably blocks biochemical activities related to arginine dihydrolase and lysine decarboxylase, but the scope of the present invention is not limited thereto. .
본 발명의 구체예에서, 상기 균주는 AMC(amoxicillin/clavulanic acid), 암피실린(ampicillin, AM), 세팔로틴(cephalothin, CF), 겐타마이신(gentamicin, GM), 카나마이신(kanamycin, K), 네오마이신(neomycin, N) 및 SAM(ampicillin/sulbactam)으로 이루어진 군에서 선택된 1종 이상의 항생제에 대해 항생제 감수성을 나타내는 것이 바람직하다. In an embodiment of the present invention, the strain is AMC (amoxicillin / clavulanic acid), ampicillin (ampicillin, AM), cephalothin (cf), gentamicin (GM), kanamycin (kanamycin, K), neo It is preferable to exhibit antibiotic sensitivity to at least one antibiotic selected from the group consisting of neomycin (N) and ampicillin/sulbactam (SAM).
본 발명의 구체예에서, 상기 균주는 flagellin FliC의 발현이 야생형 균주보다 감소되고, porin OmpD의 발현이 야생형 균주보다 증가된 것이 바람직하다.In an embodiment of the present invention, the strain preferably has a lower expression of flagellin FliC than the wild-type strain and an increased expression of porin OmpD than the wild-type strain.
본 발명의 구체예에서, 상기 균주는 숙주 내에서 비장종대(splenomegaly)를 유도하지 않는 것이 바람직하다.In an embodiment of the present invention, it is preferred that the strain does not induce splenomegaly in the host.
본 발명에 있어서, 비장종대는 비장의 용적이나 중량이 정상 범위를 넘어 증가된 것을 의미한다. 비장은 생체의 림프조직 중에서 최대의 것이기 때문에, 조혈장기의 계통적 질환이 있을때에는 생체내의 다른 조혈장기와 함께 반응하여 종대한다. 비장종대는 (1) 감염증(말라리아, 아급성심내막염, 살모넬라증 등), (2) 혈액질환(백혈병, 빈혈 등), (3) 대사이상에서 주로 나타난다.In the present invention, spleen enlargement means that the volume or weight of the spleen is increased beyond the normal range. Since the spleen is the largest of the lymphoid tissue in the body, when there is a systemic disease of the hematopoietic organ, it reacts with other hematopoietic organs in the body and expands. Splenomegaly is mainly seen in (1) infections (malaria, subacute endocarditis, salmonellosis, etc.), (2) blood diseases (leukemia, anemia, etc.), and (3) metabolic abnormalities.
본 발명의 다른 양태에 따르면, 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 포함하는, 살모넬라증(Salmonellosis) 예방 또는 치료용 백신 조성물; 살모넬라균에 대한 면역원성 조성물;을 제공한다.According to another aspect of the present invention, a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dried product of the culture A vaccine composition for preventing or treating Salmonellosis, comprising at least one member selected from the group; An immunogenic composition against Salmonella.
상기 배양물은 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주를 배양배지 또는 배양액에서 배양한 것을 의미하고, 상기 배양물은 상기 균주를 포함하는 배양물이다. 상기 배양물 또는 배양 농축물은 그 제형이 한정되지 않고, 일 예로 상기 제형은 액체 또는 고체일 수 있다. The culture means that the YjeK gene-deleted Salmonella typhimurium strain is cultured in a culture medium or culture medium, and the culture is a culture containing the strain. The formulation of the culture or culture concentrate is not limited, and for example, the formulation may be liquid or solid.
상기 배지는 특정 미생물을 배양하기 위하여, 배양대상 즉, 배양체가 되는 미생물이 필요로 하는 영양물질을 포함하는 것으로 특수한 목적을 위한 물질이 추가로 첨가되어 혼합된 것 일 수 있다. 상기 배지는 배양기 또는 배양액이라고도 할 수 있고, 천연배지, 합성배지 또는 선택배지를 모두 포함하는 개념이다. 배양한 배지로부터 분리하여 얻은 것 일 수 있고, 상기 배지는 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주가 성장할 수 있는 것이라면 한정되지 않고 이용할 수 있다. In order to cultivate a specific microorganism, the medium contains nutrients required by the microorganism to be cultured, that is, the microorganism to be cultured, and may be a mixture in which a substance for a special purpose is additionally added. The medium may also be referred to as an incubator or a culture medium, and is a concept that includes all of a natural medium, a synthetic medium, or a selective medium. It may be obtained by separating from the cultured medium, and the medium may be used without limitation as long as the YjeK gene-deleted Salmonella typhimurium strain can grow.
상기 배지는 일 예로, 보통 한천배지(또는 영양 한천배지; Nutrient agar), TSA(tryptic soy agar) 배지, 표준한천배지(Standard Methods Agar; Plate Count Agar), 유당배지(lactose Broth), BGlB 배지(Brilliant Green lactose Bile Broth), 2배 농도 BGlB 배지, Endo 한천배지(Endo Agar), EMB 한천배지(Eosin methylene blue agar), 보통 배지(또는 영양 배지; Nutrient Broth), 데스옥시콜레이트 유당 한천배지(Desoxycholate lactose Agar), LB 배지(Luria-bertani Broth) 또는 EC 배지(EC Broth)일 수 있다. The medium is, for example, normal agar medium (or nutrient agar medium; Nutrient agar), TSA (tryptic soy agar) medium, standard agar medium (Standard Methods Agar; Plate Count Agar), lactose medium (lactose broth), BGlB medium ( Brilliant Green lactose Bile Broth), double strength BGlB medium, Endo agar medium (Endo agar), EMB agar medium (Eosin methylene blue agar), normal medium (or nutrient medium; Nutrient Broth), desoxycholate lactose agar medium (Desoxycholate medium) lactose Agar), LB medium (Luria-bertani Broth) or EC medium (EC Broth).
상기 조성물은 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주의 안정적인 제제화를 목적으로, 수화제, 입제 또는 캡슐제로 제제화하여 사용할 수 있고, 상기 종류에 한정되는 것은 아니다.For the purpose of stable formulation of the YjeK gene-deleted Salmonella typhimurium strain, the composition may be formulated and used as a wettable powder, granule or capsule, but is not limited to the above type.
본 발명의 수화제는 균주를 접종한 고체배지를 건조시켜 분쇄한 후 계면활성제, 증량제 또는 영양제를 첨가하여 혼합하여 제조할 수 있다. 상기 계면활성제로는 폴리카복실레이트, 소듐 리그노설포네이트, 칼슘 리그노설포네이트, 소듐 디알킬 설포석시네이트, 소듐 알킬 아릴 설포네이트, 폴리옥시에틸렌 알킬 페닐 에테르, 소듐트리폴리포스페이트, 폴리옥시에틸렌 알킬 아릴 포스포릭 에스테르, 폴리옥시에틸렌 알킬 아릴 에테르, 폴리옥시에틸렌 알킬 아릴 폴리머, 소듐 설포네이트 나프탈렌 포름알데히드, 트리톤 100 및 트윈 80으로 이루어진 군으로부터 선택되는 하나 또는 둘 이상을 사용하며, 증량제 및 영양제로는 콩가루, 쌀, 밀, 황토, 규조토, 덱스트린 (dextrin), 포도당 및 전분으로 이루어진 군으로부터 선택되는 하나 또는 둘 이상을 사용할 수 있다.The hydrating agent of the present invention may be prepared by drying and pulverizing the solid medium inoculated with the strain, and then adding and mixing a surfactant, an extender or a nutrient. As the surfactant, polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene alkyl One or two or more selected from the group consisting of aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80 are used, and the extenders and nutrients include One or two or more selected from the group consisting of soy flour, rice, wheat, ocher, diatomaceous earth, dextrin, glucose, and starch may be used.
또한, 본 발명의 입제는 균주를 접종한 고체배지를 건조시켜 분쇄한 후 계면활성제, 증량체 및 영양제는 상기 기재된 것과 동일한 것을 사용할 수 있다. 또한, 본 발명의 입제는 균주의 표면 활성제, 비활성 담체, 보존제, 습윤제, 공급촉진제, 유인제, 캡슐화제, 결합제, 유화제, 염료, UV 보호제, 완충제 및 흐름제로 이루어진 군으로부터 선택되는 하나 또는 둘 이상의 것을 추가로 첨가하여 제조할 수 있다.In addition, the granules of the present invention may use the same surfactants, extenders and nutrients as described above after drying and pulverizing the solid medium inoculated with the strain. In addition, the granule of the present invention is one or two or more selected from the group consisting of a surface active agent of the strain, an inert carrier, a preservative, a wetting agent, a supply promoter, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protective agent, a buffer and a flow agent It can be prepared by adding more.
본 발명의 용어 “예방”이란 조성물의 투여로 질병을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.The term "prevention" of the present invention means any action that suppresses or delays the onset of a disease by administering a composition.
본 발명의 용어 “치료”란 조성물의 투여로 상기 질병의 증세가 호전되거나 상기 질병의 억제 또는 경감 및 이롭게 변경되는 모든 행위를 의미한다.The term "treatment" of the present invention refers to all activities that improve the symptoms of the disease or inhibit or alleviate the disease and change beneficially by the administration of the composition.
본 발명의 구체예에서, 상기 조성물은 복강내 투여, 정맥내 투여, 근육내 투여, 비강내 투여, 관절내(intra-articular) 투여, 활액내(intra-synovial) 투여, 수막강내 투여, 간내(intrahepatic) 투여, 병변내(intralesional) 투여 또는 두개강내(intracranial) 투여되는 것이 바람직하며, 더 바람직하게는 복강내 투여되는 것일 수 있다.In an embodiment of the present invention, the composition is intraperitoneal administration, intravenous administration, intramuscular administration, intranasal administration, intra-articular administration, intra-synovial administration, intrathecal administration, intrahepatic administration (intrahepatic) administration, intralesional administration, or intracranial administration is preferable, and more preferably, it may be intraperitoneal administration.
본 발명에 있어서, 백신은 항원 물질을 포함하는 수의학용 백신으로, 살모넬라균에 대하여 특이적이고 능동 또는 수동의 면역성을 유도하기 위한 목적으로 투여된다. 본 발명의 실시예에서는 본 발명의 살모넬라균 감염을 효과적으로 방어한다는 것을 확인한바, 살모넬라증의 예방에 유용하게 사용될 수 있다.In the present invention, the vaccine is a veterinary vaccine containing an antigenic material, which is specific for Salmonella and is administered for the purpose of inducing active or passive immunity. In the examples of the present invention, it was confirmed that the present invention effectively protects against Salmonella infection, and thus it can be usefully used for the prevention of salmonellosis.
본 발명에 따른 백신 조성물의 투여는 면역학적 유효량으로 투여할 수 있다. 상기 "면역학적 유효량"이란 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주가 관련된 질병의 예방 효과를 나타낼 수 있을 정도의 충분한 양과 부작용이나 심각한 또는 과도한 면역반응을 일으키지 않을 정도의 양을 의미하며, 정확한 투여 농도는 투여될 특정 면역원에 따라 달라지며, 예방 접종 대상의 연령, 체중, 건강, 성별, 개체의 약물에 대한 민감도, 투여 경로, 투여 방법 등 의학 분야에 잘 알려진 요소에 따라 당업자에 의해 용이하게 결정될 수 있으며, 1회 내지 수회 투여할 수 있다.Administration of the vaccine composition according to the present invention can be administered in an immunologically effective amount. The "immunologically effective amount" means an amount sufficient to exhibit a preventive effect against a disease associated with a YjeK gene-deleted strain of Salmonella typhimurium and an amount sufficient to not cause side effects or serious or excessive immune reactions, and is administered accurately. The concentration varies depending on the specific immunogen to be administered, and can be easily determined by a person skilled in the art according to factors well known in the medical field, such as the age, weight, health, sex of the subject to be vaccinated, sensitivity to drugs of the individual, administration route, and administration method. It can be administered once or several times.
본 발명에 따른 백신 조성물은 유효 성분인 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주 이외에도 백신 조성물을 구성하는 데 적절한 하나 이상의 면역 증강제 또는 부형제 또는 담체를 포함할 수 있다.The vaccine composition according to the present invention may include, in addition to the YjeK gene-deleted Salmonella typhimurium strain as an active ingredient, one or more immune enhancers or excipients or carriers suitable for constituting the vaccine composition.
본 발명의 백신 조성물에 포함될 수 있는 면역증강제는 주사한 동물의 면역 반응을 증대시키는 물질을 의미하는 것으로, 다수의 상이한 면역증강제가 기술 분야의 당업자에게 공지되어 있다. 상기 면역증강제는 프로인트 완전 및 불완전 면역 증강제, 비타민 E, 비이온성 차단 폴리머, 뮤라밀디펩티드, Quil A, 광유 및 무광물유 및 카보폴(Carbopol), 유중수형 유제 면역증강제 등을 포함하며, 이에 제한되는 것은 아니다.An adjuvant that may be included in the vaccine composition of the present invention refers to a substance that enhances the immune response of an injected animal, and many different adjuvants are known to those skilled in the art. The immune enhancers include Freund's complete and incomplete immune enhancers, vitamin E, nonionic blocking polymers, muramyl dipeptide, Quil A, mineral oil and non-mineral oil and Carbopol, water-in-oil emulsion immune enhancers, etc., but are limited thereto it is not going to be
본 발명의 백신 조성물에 포함될 수 있는 담체는 기술 분야의 당업자에게 공지되어 있으며, 단백질, 설탕 등을 포함하지만, 이로 제한되는 것은 아니다. 상기의 담체는 수용액 또는 비-수용액, 현탁액, 및 에멀전일 수 있다. 비-수용액 담체의 예는 프로필렌 글리콜, 폴리에틸렌 글리콜, 식용유 예컨대 올리브 오일, 및 주사 가능한 유기 에스테르 예컨대 에틸 올리에이트를 들 수 있다. 수용액 담체는 식염수 및 완충배지를 포함하는, 물, 알콜/수용액, 에멀전 또는 현탁액을 포함한다. 비경구 담체는 염화나트륨 용액, 링거 덱스트로스, 덱스트로스 및 염화나트륨, 유산처리 링거 또는 고정 오일을 포함한다. 정맥주사용 담체는 예컨대 링거 덱스트로스를 기본으로 하는 것과 같은 전해질 보충제, 액체 및 영양 보충제 등을 포함한다.Carriers that may be included in the vaccine composition of the present invention are known to those skilled in the art, and include, but are not limited to, proteins, sugars, and the like. The above carriers may 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 carriers include water, alcoholic/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. Carriers for intravenous injection include electrolyte replenishers, liquid and nutritional supplements, and the like, such as those based on Ringer's dextrose.
본 발명의 백신 조성물은 방부제 및 기타 첨가제 예컨대 예를 들면 항미생물제제, 항산화제, 킬레이트제, 불활성 가스 등과 같은 것을 추가로 포함할 수 있다. 상기 방부제는 포르말린, 티메로살, 네오마이신, 폴리믹신 B 및 암포테리신 B 등을 포함한다. 본 발명의 백신 조성물은 하나 이상의 적절한 유화제, 예로서 스판(Span) 또는 트윈(Tween)을 포함할 수 있다. 또한 본 발명의 백신 조성물은 보호제를 포함할 수 있으며, 당업계에 공지된 보호제를 제한 없이 사용할 수 있고, 이는 락토오스(Lactose; LPGG) 또는 트레할로오스(Trehalose; TPGG)를 포함할 수 있으나, 이에 제한되는 것은 아니다.The vaccine composition of the present invention may further contain preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like. The preservatives include formalin, thimerosal, neomycin, polymyxin B and amphotericin B, and the like. The vaccine composition of the present invention may include one or more suitable emulsifiers, such as Span or Tween. In addition, the vaccine composition of the present invention may include a protecting agent, and a protecting agent known in the art may be used without limitation, which may include lactose (LPGG) or trehalose (TPGG), It is not limited thereto.
본 발명의 또 다른 양태에 따르면, 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 포함하는 살모넬라증 예방 또는 개선용 사료 조성물; 또는 살모넬라증 예방 또는 개선용 사료 첨가제 조성물;일 수 있다.According to another aspect of the present invention, a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dried form of the culture A feed composition for preventing or improving salmonellosis comprising at least one member selected from the group consisting of; Or a feed additive composition for preventing or improving salmonellosis; it may be.
본 발명에 있어서, 사료는 동물이 먹는 임의의 천연 또는 인공 구성식, 한끼식 등 또는 상기 한끼식의 성분을 의미하며, 본 발명에 따른 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주를 유효성분으로 포함하는 사료는 당업계에 공지된 다양한 형태의 사료로 제조 가능하며, 바람직하게는 농후 사료, 조사료 및/또는 특수사료가 포함될 수 있으나, 이로 제한되는 것은 아니다.In the present invention, feed means any natural or artificial composition, one meal, etc., or a component of the one meal meal eaten by animals, and includes the YjeK gene-deleted Salmonella typhimurium strain according to the present invention as an active ingredient. The feed may be prepared with various types of feed known in the art, and preferably may include concentrated feed, roughage and / or special feed, but is not limited thereto.
본 발명에 있어서, 사료 첨가제는 동물에 있어 질병 증상의 완화, 영양소 보충 및 체중감소 예방, 사료 내 섬유소의 소화 이용성 증진, 유질개선, 번식장애 예방 및 수태율 향상, 하절기 고온 스트레스 예방 등 다양한 효과를 목적으로 사료에 첨가하는 물질을 의미한다.In the present invention, the feed additive is aimed at various effects such as alleviation of disease symptoms in animals, nutrient supplementation and weight loss prevention, improvement of digestibility of fiber in feed, oil quality improvement, reproduction disorder prevention and conception rate improvement, and summer high temperature stress prevention. means a substance added to feed.
본 발명의 사료 조성물 및 사료 첨가제 조성물은 사료관리법상의 보조사료에 해당하며, 탄산수소나트륨, 벤토나이트(bentonite), 산화마그네슘, 복합광물질 등의 광물질제제, 아연, 구리, 코발트, 셀레늄 등의 미량 광물질인 미네랄제제, 케로틴, 비타민 A, D, E, 니코틴산, 비타민 B 복합체 등의 비타민제, 메티오닌, 라이신 등의 보호아미노산제, 지방산 칼슘염 등의 보호지방산제, 생균제(유산균제), 효모배양물, 곰팡이 발효물 등의 생균, 효모제 등이 추가로 포함될 수 있다.The feed composition and feed additive composition of the present invention correspond to supplementary feed under the Feed Control Act, and include mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, and trace minerals such as zinc, copper, cobalt, and selenium. Mineral preparations, vitamins such as kerotene, vitamins A, D, E, nicotinic acid, and vitamin B complex, protective amino acids such as methionine and lysine, protective fatty acids such as calcium salts of fatty acids, probiotics (lactic acid bacteria), yeast cultures, molds Live bacteria such as fermentation products, yeast agents, and the like may be further included.
상기 사료 중 농후사료로는 밀, 귀리, 옥수수 등의 곡류를 포함하는 종자열매류, 곡물을 정제하고 얻는 부산물로서 쌀겨, 밀기울, 보릿겨 등을 포함하는 겨류, 콩, 유체, 깨, 아마인, 코코야자 등을 채유하고 얻는 부산물인 깻묵류와 고구마, 감자 등에서 녹말을 뺀 나머지인 녹말찌꺼기의 주성분인 잔존녹말질류 등의 찌꺼기류, 어분, 물고기찌꺼기, 어류에서 얻은 신선한 액상물을 농축시킨 것인 피시솔루블(fish soluble), 육분, 혈분, 우모분, 탈지분유, 우유에서 치즈, 탈지유에서 카제인을 제조할 때의 잔액인 훼이(whey)를 건조한 건조훼이 등의 동물질사료, 효모, 클로렐라, 해조류가 있으나 이에 제한되지 않는다.Among the feeds, the enriched feed includes seed fruits including grains such as wheat, oats and corn, bran including rice bran, bran, barley bran, etc. as a by-product obtained by refining grains, soybeans, fluids, sesame seeds, linseed, coco Fish meal, which is a by-product obtained from oil extraction of palms, residual starch, which is the main component of starch residue, which is the remainder after removing starch from sweet potatoes, potatoes, etc., fish meal, fish residue, and fresh liquid obtained from fish Fish soluble, meat meal, blood meal, feather meal, skim milk powder, animal feed such as dry whey, yeast, chlorella, seaweed but not limited thereto.
상기 사료 중 조사료로는 야초, 목초, 풋베기 등의 생초사료, 사료용 순무, 사료용 비트, 순무의 일종인 루터베어거 등 의 뿌리채소류, 생초, 풋베기작물, 곡실 등을 사일로에 채워 놓고 젖산발효시킨 저장사료인 사일리지(silage), 야초, 목초를 베어 건조시킨 건초, 종축용 작물의 짚, 콩과 식물의 나뭇잎이 있으며, 이에 제한되지 않는다. 특수사료에는 굴껍데기, 암염 등의 미네랄 사료, 요소나 그 유도체인 디우레이드이소부탄 등의 요소사료, 천연사료원료만을 배합했을 때 부족하기 쉬운 성분을 보충하거나, 사료의 저장성을 높이기 위해서 배합사료에 미량으로 첨가하는 물질인 사료첨가물, 식이보조제가 있으나 이에 제한되지 않는다.Forage among the feed includes grass feed such as wild grass, grass, green cutting, turnip for feed, beet for feed, root vegetables such as Lutherbearer, a type of turnip, raw grass, green crops, grains, etc. are filled in a silo and fermented with lactic acid It includes, but is not limited to, silage, which is a stored feed, grass, hay made by cutting down grass, straw of breeding crops, and leaves of leguminous plants. Special feeds include mineral feeds such as oyster shells and rock salt, urea feeds such as urea or its derivative, diureide isobutane, and supplements for ingredients that are likely to be insufficient when only natural feed ingredients are mixed, or formulated feeds to improve the storability of feeds. There are feed additives and dietary supplements, which are substances added in small amounts, but are not limited thereto.
본 발명의 또 다른 양태에 따르면, 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 이를 필요로 하는 개체에 투여하는 단계;를 포함하는 살모넬라증의 예방 또는 치료방법을 제공한다.According to another aspect of the present invention, a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dried form of the culture It provides a method for preventing or treating salmonellosis comprising; administering at least one selected from the group consisting of to a subject in need thereof.
본 발명의 구체예에서, 상기 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주는 정맥내 투여, 근육내 투여, 비강내 투여, 관절내(intra-articular) 투여, 활액내(intra-synovial) 투여, 수막강내 투여, 간내(intrahepatic) 투여, 병변내(intralesional) 투여 또는 두개강내(intracranial) 투여되는 것이 바람직하고, 더 바람직하게는 근육내 또는 비강내 투여되는 것이나, 이에 제한되지 않는다.In an embodiment of the present invention, the YjeK gene-deleted Salmonella typhimurium strain is intravenous administration, intramuscular administration, intranasal administration, intra-articular administration, intra-synovial administration, water Intrathecal administration, intrahepatic administration, intralesional administration or intracranial administration is preferred, and intramuscular or intranasal administration is more preferred, but is not limited thereto.
본 발명의 살모넬라증의 예방 또는 치료방법은 YjeK 유전자가 결실된 살모넬라 티피뮤리움 균주의 투여로 인해 생체 내 면역 반응을 증가시키는 것이 바람직하며, 보다 상세하게는 항체가, 면역반응 관련 인자의 발현을 조절하는 것이 바람직하다.In the method for preventing or treating salmonellosis of the present invention, it is preferable to increase the immune response in vivo by administering a Salmonella typhimurium strain in which the YjeK gene is deleted, and more specifically, the antibody regulates the expression of factors related to the immune response. It is desirable to do
중복되는 내용은 본 명세서의 복잡성을 고려하여 생략하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
[실험예][Experimental example]
실험예 1. 박테리아 균주, 플라스미드 및 성장 조건Experimental Example 1. Bacterial strains, plasmids and growth conditions
이 연구에 사용된 박테리아 균주 및 플라스미드는 표 1과 같다.Bacterial strains and plasmids used in this study are listed in Table 1.
| Strainsstrains | Relevant characteristicsRelevant characteristics | ReferencesReferences |
| ST1120ST1120 | Wild-type S. Typhimurium isolated in KoreaWild-type S. Typhimurium isolated in Korea | Kim et al., 2017Kim et al., 2017 |
| ST2160ST2160 | ST1120 derivative(ΔyjeK::FRT, CmR)ST1120 derivative(Δ yjeK ::FRT, Cm R ) | 본 발명the present invention |
| ST2161ST2161 | ST2160 derivative(ΔyjeK)ST2160 derivative( ΔyjeK ) | 본 발명the present invention |
| ST14028ST14028 | ATCC14028ATCC14028 | ATCCATCC |
| ST2173ST2173 | ST14028 with plasmid pBBR1-MCS4(AMR)ST14028 with plasmid pBBR1-MCS4 (AM R ) | 본 발명the present invention |
| PlasmidsPlasmids | CharacteristicsCharacteristics | |
| pTP233pTP233 | λ-Red recombinase, temperature sensitive replication(TER)λ-Red recombinase, temperature sensitive replication (TE R ) | Poteete and Fenton, 1984Poteete and Fenton, 1984 |
| pKD3pKD3 | FRT-Cm-FRT cassette(CbRCmR)FRT-Cm-FRT cassette (Cb R Cm R ) | Datsenko and Wanner, 2000Datsenko and Wanner, 2000 |
| pCP20pCP20 | FLP recombinase, temperature sensitive replication(AMR)FLP recombinase, temperature sensitive replication (AM R ) | Cherepanov and Wackernagel, 1995Cherepanov and Wackernagel, 1995 |
| pBBR1-MCS4pBBR1-MCS4 | Broad-host-range cloning vector(AMR)Broad-host-range cloning vector (AM R ) | Truong, et al. 2014Truong, et al. 2014 |
모든 실험에서 대조군으로 국내에서 분리된 야생형 Salmonella enterica serovar Typhimurium 1120(ST1120)을 사용하였다. 동물 챌린지 실험에서 플라스미드 pBBR1-MCS4로 형질전환된 S. Typhimurium 14028 균주(ST2173)를 사용하였다. 상기 ST2173 균주는 암피실린 내성 유전자를 포함한다. 박테리아 균주는 달리 명시되지 않는 한 필요한 경우 150μg/ml의 암피실린(Duchefa)과 함께 37°C의 Luria-Bertani(LB) broth(Duchefa, Haarlem, 네덜란드)에서 배양되었다. 모든 항생제는 Sigma-Aldrich(St. Louis, MO, USA)에서 구입했다. API 20E 키트(bioMerieux, Inc., Durham, NC, USA)를 사용하여 박테리아의 생화학적 특성을 확인했다. In all experiments, as a control, wild-type Salmonella enterica serovar Typhimurium 1120 (ST1120) isolated in Korea was used. In animal challenge experiments, S. Typhimurium 14028 strain (ST2173) transformed with the plasmid pBBR1-MCS4 was used. The ST2173 strain contains an ampicillin resistance gene. Bacterial strains were cultured in Luria-Bertani (LB) broth (Duchefa, Haarlem, The Netherlands) at 37°C with 150 μg/ml ampicillin (Duchefa) when necessary unless otherwise specified. All antibiotics were purchased from Sigma-Aldrich (St. Louis, MO, USA). The biochemical properties of the bacteria were confirmed using the API 20E kit (bioMerieux, Inc., Durham, NC, USA).
yjeK 유전자가 결실된 Δ yjeK 돌연변이 균주는 플라스미드 pTP233, pKD3 및 pCP20을 사용한 λ red 재조합 방법을 사용하여 부위 지정 돌연변이 유발을 통해 생성되었다. 구체적으로, pTP233을 포함하는 ST1120은 yjeK 유전자와 상동인 서열이 측면에 있는 클로람페니콜 내성 유전자 카세트로 형질전환하여 ST2160 균주를 제조하였다. ST2160 균주에서 염색체 yjeK 및 CM 내성 카세트 사이의 상동 재조합은 40 μg/mL 클로람페니콜을 포함하는 LB 한천에 플레이팅한 후 진단 중합효소 연쇄 반응을 통해 확인하였다. pCP20 플라스미드 도입 후 클로람페니콜 감수성 균주를 선택하고, 염색체의 클로람페니콜 저항성 유전자를 제거하여 ST2161 균주를 얻었다. 마지막으로, 42℃에서 클로람페니콜-감수성 균주를 배양하여 pCP20 플라스미드를 제거하고, yjeK 및 CM 카세트의 결실을 진단 PCR을 사용하여 추가로 검증하였다.A ΔyjeK mutant strain in which the yjeK gene was deleted was generated through site-directed mutagenesis using the λ red recombination method using plasmids pTP233, pKD3 and pCP20. Specifically, ST1120 containing pTP233 was transformed with a chloramphenicol resistance gene cassette flanked by sequences homologous to the yjeK gene to prepare ST2160 strain. Homologous recombination between the chromosome yjeK and the CM resistance cassette in strain ST2160 was confirmed by diagnostic polymerase chain reaction after plating on LB agar containing 40 μg/mL chloramphenicol. After introducing the pCP20 plasmid, a chloramphenicol-sensitive strain was selected, and the chloramphenicol-resistance gene was removed from the chromosome to obtain strain ST2161. Finally, the pCP20 plasmid was removed by culturing the chloramphenicol-sensitive strain at 42° C., and deletion of the yjeK and CM cassettes was further verified using diagnostic PCR.
균주 구성 및 검증에 사용된 모든 프라이머는 표 2와 같다.All primers used for strain construction and verification are shown in Table 2.
| PrimersPrimers | Sequence(5’-3’)Sequence(5’-3’) |
| yjeK_long F yjeK_long F | TTAGCACTGCCGAAGCTGTAAATCCAGCGGTGTTTTGCTTGTGTAGGCTGGAGCTGCTTCGAAGTTAGCACTGCCGAAGCTGTAAATCCAGCGGTGTTTTGCTTGTGTAGGCTGGAGCTGCTTCGAAG |
| yjeK_long R yjeK _long R | GATGAAAACCTCCGGGCAGGACAGGACGCCAGGCGCTTGTTCATATGAATATCCTCCTTAGTTCCTGATGAAAACCTCCGGGCAGGACAGGACGCCAGGCGCTTGTTCATATGAATATCCTCCTTAGTTCCT |
| yjeK_com F yjeK_com F | ATTAGCACTGCCGAAGCTGTAATTAGCACTGCCGAAGCTGTA |
| yjeK_com R yjeK _com R | CGAGAAGATTGGTTAGCGCAACCGAGAAGATTGGTTAGCGCAAC |
| hilA qPCR F hilA qPCR F | GCTGCACCAGGAAAGCATTAAGGCTGCACCAGGAAAGCATTAAG |
| hilA qPCR R hilA qPCR R | CGAAGTCCGGGAATACATCTGACGAAGTCCGGGAATACATCTGA |
| hilC qPCR F hilC qPCR F | GCCGCTGAAGAGGTGAGTTTTAGCCGCTGAAGAGGTGAGTTTTA |
| hilC qPCR R hilC qPCR R | AATATTTCCAGCCCCCATACGAATATTTCCAGCCCCCCATACG |
| hilD qPCR F hilD qPCR F | GCTGTTCCTGCTTACTGCTTTTCGCTGTTCCTGCTTACTGCTTTTTC |
| hilD qPCR R hilD qPCR R | AATGTTGTAAACGCGCTCCTTTAATGTTGTAAACGCGCTCCTTT |
| invA qPCR F invA qPCR F | ACAAAACATATGCTGGACCAACTGACAAAACATATGCTGGACCAACTG |
| invA qPCR R invA qPCR R | ACGCTGCAAAACTTCAGATATACGACGCTGCAAAACTTCAGATATACG |
| invF qPCR F invF qPCR F | GCGGAAAAGCGAAGAGTGAAGCGGAAAAGCGAAGAGTGAA |
| invF qPCR R invF qPCR R | AACGGCTAATTGGGTGATGTTCAACGGCTAATTGGGTGATGTTC |
| sipC qPCR F sipC qPCR F | CGAAGGGATGAATGCGTTGTCGAAGGGATGAATGCGTTGT |
| sipC qPCR R sipC qPCR R | GCAGCCCCTTATATTCCAGTTTGGCAGCCCCTTATATTCCAGTTTG |
| sopA qPCR F sopA qPCR F | TACGTCACAAAGCCAACCTCTCTTACGTCACAAAGCCAACCTCTCT |
| sopA qPCR R sopA qPCR R | GTGGCATTTGCAGCCAGATAGTGGCATTTGCAGCCAGATA |
| sopB qPCR F sopB qPCR F | GCAATGATTTACGCCCTGAAGGCAATGATTTACGCCCTGAAG |
| sopB qPCR R sopB qPCR R | TGGCGCGCTAATGTATTGACTGGCGCGCTAATGTATTGAC |
| sopE2 qPCR F sopE2 qPCR F | CGGAGAAGGACATTTTTGCAACGGAGAAGGACATTTTTGCAA |
| sopE2 qPCR R sopE2 qPCR R | TGGGTAGTCGGTATGTCTGTTTGTTGGGTAGTCGGTATGTCTGTTTGT |
| sptP qPCR F sptP qPCR F | GGCGATACAAAAGTGGCAGAAGGCGATACAAAAGTGGCAGAA |
| sptP qPCR R sptP qPCR R | CGCAGGCTATTTCCATCCATCGCAGGCTATTTCCATCCAT |
| ssaD qPCR F ssaD qPCR F | GGTGGAATGGGTGTCCTGTTAAGGTGGAATGGGTGTCCTGTTAA |
| ssaD qPCR R ssaD qPCR R | CGCCCGCACATAATGAATATTCGCCCGCACATAATGAATATT |
| ssaK qPCR F ssaK qPCR F | GAGGCATTGATGCGAGAAACTGAGGCATTGATGCGAGAAACT |
| ssaK qPCR R ssaK qPCR R | CGGCATATCGTGTTGAGGAACGGCATATCGTGTTGAGGAA |
| ssaQ qPCR F ssaQ qPCR F | CATCCTGCAGTAATCTACCACATCACATCCTGCAGTAATCTACCACATCA |
| ssaQ qPCR R ssaQ qPCR R | AAACCCGTTGGCCATGTAAAAAACCCGTTGGCCATGTAAA |
| sseD qPCR F sseD qPCR F | ATGCGCAGCTATAACGTAGAAAAAATGCGCAGCTATAACGTAGAAAAA |
| sseD qPCR R sseD qPCR R | TGCTCTAAACGCTTCATCAATTGTGCTCTAAACGCTTCATCAATTG |
| sseF qPCR F sseF qPCR F | GGTTGCTGCAGCGGTAATTTGGTTGCTGCAGCGGTAATTT |
| sseF qPCR R sseF qPCR R | CACAGCAAGCATCCCCAATACACAGCAAGCATCCCCAATA |
| ssrA qPCR F ssrA qPCR F | GGTTCAGAAACGGCAGCATATAAGGTTCAGAAACGGCAGCATATAA |
| ssrA qPCR R ssrA qPCR R | GTTCAGCTTCTTCAAACCGTTGAGTTCAGCTTCTTCAAACCGTTGA |
| ssrB qPCR F ssrB qPCR F | TCTGTTAGCGGCATTGCAAATCTGTTAGCGGCATTGCAAA |
| ssrB qPCR R ssrB qPCR R | GGTCGTGTCAGCGTTTAATTCAGGTCGTGTCAGCGTTTAATTCA |
| yjeK qPCR F yjeK qPCR F | ACTGGTCGCCCGCTTTGACTGGTCGCCCGCTTTG |
| yjeK qPCR R yjeK qPCR R | CGCCTCGTCCACTTCATTAGCGCCTCGTCCACTTCATTAG |
| efp qPCR F efp qPCR F | CGATTTTCGTTCCGGTCTTAACGATTTTCGTTCCGGTCTTAA |
| efp qPCR R efp qPCR R | CCTGGCCTTTACCCGGTTTCCTGGCCTTTACCCGGTTT |
| yjeA qPCR F yjeA qPCR F | AAAACGTGCGGCGATTATGAAAACGTGCGGCGATTATG |
| yjeA qPCR R yjeA qPCR R | ACTCATGCAGGGCGTCTCAACTCATGCAGGGCGTCTCA |
| gyrB qPCR F gyrB qPCR F | TCGCTCAGCAGTTCGTTCATTCGCTCAGCAGTTCGTTCAT |
| gyrB qPCR R gyrB qPCR R | GATTGCGGTGGTTTCCGTAAGATTGCGGTGGTTTCCGTAA |
실험예 2. 항생제 감수성 검사Experimental Example 2. Antibiotic susceptibility test
Mueller-Hinton agar(Becton Dickinson, Sparks, MD, USA)에 대한 디스크 확산 테스트를 사용하여 박테리아 항생제 감수성을 평가했다. 박테리아 세포는 LB broth를 이용하여 온도 37°C 16-18시간 동안 배양하였다. 배양된 세포의 600 nm에서 광학 밀도(OD600)가 0.6-0.7이었으며, 배양된 세포는 5 × 107 cells 에 해당했다. 집락 형성 단위(colony forming units, CFU)/mL를 항생제 디스크(BBL™ Sensi-Disc™, Becton Dickinson)를 배치하기 전에 멸균 면봉을 사용하여 Mueller-Hinton(MH) 한천에 도말했다. 상기 항생체 디스크를 이용하여, 항생제인 AMC(amoxicillin/clavulanic acid) 30μg, 암피실린(ampicillin, AM) 10μg, 세팔로틴(cephalothin, CF) 30μg, 겐타마이신(gentamicin, GM) 10μg, 카나마이신(kanamycin, K) 30μg, 날리딕산(nalidixic acid, NA) 30μg, 네오마이신(neomycin, N) 30μg, SAM(ampicillin/sulbactam) 20μg, SXT(sulfamethoxazole/trimethoprim) 25μg 및 테트라사이클린(tetracyclineTE) 30μg에 대한 항생제 감수성을 확인하였으며, 성장 억제 구역의 직경은 항생제 감수성을 결정하는 데 사용되었다.Bacterial antibiotic susceptibility was assessed using the disk diffusion test on Mueller-Hinton agar (Becton Dickinson, Sparks, MD, USA). Bacterial cells were incubated in LB broth at 37°C for 16-18 hours. The optical density (OD 600 ) at 600 nm of the cultured cells was 0.6-0.7, and the number of cultured cells corresponded to 5 × 10 7 cells. Colony forming units (CFU)/mL were smeared on Mueller-Hinton (MH) agar using sterile swabs prior to placement of antibiotic discs (BBL™ Sensi-Disc™, Becton Dickinson). Using the antibiotic disc, 30 μg of antibiotics AMC (amoxicillin/clavulanic acid), 10 μg of ampicillin (AM), 30 μg of cephalothin (CF), 10 μg of gentamicin (GM), kanamycin, K) Antibiotic susceptibility to 30 μg, nalidixic acid (NA) 30 μg, neomycin (N) 30 μg, SAM (ampicillin/sulbactam) 20 μg, SXT (sulfamethoxazole/trimethoprim) 25 μg and tetracycline TE 30 μg was identified, and the diameter of the growth inhibition zone was used to determine antibiotic susceptibility.
실험예 3. 운동성 테스트Experimental Example 3. Motility test
박테리아 수영 운동성은 이전에 자세히 설명된 대로 37°C에서 반고체-한천 플레이트(0.3% 한천)에서 평가하였다. OD 600이 0.6-0.7인 각 균주를 반고체 한천에 스팟팅하고 37°C에서 배양했다. 수영 후광(swimming halo)의 직경은 각각 4시간 및 12시간에 측정되었다. 모든 실험은 세 번 반복하였다.Bacterial swimming motility was assessed on semi-solid-agar plates (0.3% agar) at 37 °C as previously detailed. Each strain with an OD 600 of 0.6–0.7 was spotted on semi-solid agar and incubated at 37°C. The diameter of the swimming halo was measured at 4 and 12 hours, respectively. All experiments were repeated three times.
실험예 4. 리포폴리사카라이드(Lipopolysaccharide, LPS) 프로파일 분석Experimental Example 4. Lipopolysaccharide (LPS) profile analysis
살모넬라 균주는 LB borth 배지에서 16시간 동안 배양하였고, LPS는 hot phenol-water method에 따라 추출하였다. 박테리아 세포를 10,000 xg에서 20분 동안 원심분리하여 수확했다. 박테리아 세포 펠렛을 용해 용액(0.1 M sodium dodecyl sulfate, 50 mM Tris base, 0.128 M NaOH)에 현탁하고 5분 동안 배양하고 P/C/I 용액(phenol:chloroform:isoamyl alcohol=25:24:1)로 처리했다. 65°C에서 15분 동안 가열한 후, 분리된 수성상을 새 바이알로 옮기고, 0.5mL의 3M Na-acetate(pH 5.6) 및 17mL의 95% 얼음처럼 차가운 에탄올과 혼합했다. LPS 분획을 -20°C에서 밤새 침전시키고, 10,000 xg로 원심분리했다. 20분 동안 침전된 LPS를 멸균수에 재현탁하고 DNase(Sigma-Aldrich) 및 RNase(Promega, Madison, WI, USA)로 37℃에서 1시간 동안 처리하였다. LPS 정제를 위해 LPS 용액을 P/C/I 용액과 혼합하고 10,000 xg에서 20분 동안 원심분리하였다. 정제된 LPS를 멸균수에 현탁하고 Pierce LAL 발색 내독소 정량 키트(Thermo Scientific Inc., IL, USA)를 사용하여 정량화했다. 5 μg의 LPS 샘플을 12% DOC-PAGE(deoxycholate-polyacrylamide gel electrophoresis) 겔에 로딩하고 LPS 프로파일을 시각화했다.Salmonella strains were cultured in LB borth medium for 16 hours, and LPS was extracted according to the hot phenol-water method. Bacterial cells were harvested by centrifugation at 10,000 xg for 20 minutes. Bacterial cell pellets were suspended in lysing solution (0.1 M sodium dodecyl sulfate, 50 mM Tris base, 0.128 M NaOH), incubated for 5 minutes, and in P/C/I solution (phenol:chloroform:isoamyl alcohol=25:24:1) treated with After heating at 65 °C for 15 min, the separated aqueous phase was transferred to a new vial and mixed with 0.5 mL of 3M Na-acetate (pH 5.6) and 17 mL of 95% ice-cold ethanol. The LPS fraction was precipitated overnight at -20 °C and centrifuged at 10,000 xg. LPS precipitated for 20 minutes was resuspended in sterile water and treated with DNase (Sigma-Aldrich) and RNase (Promega, Madison, WI, USA) at 37°C for 1 hour. For LPS purification, the LPS solution was mixed with the P/C/I solution and centrifuged at 10,000 xg for 20 minutes. Purified LPS was suspended in sterile water and quantified using the Pierce LAL Chromogenic Endotoxin Quantification Kit (Thermo Scientific Inc., IL, USA). A 5 μg LPS sample was loaded on a 12% DOC-PAGE (deoxycholate-polyacrylamide gel electrophoresis) gel and the LPS profile was visualized.
실험예 5. 외막 단백질(Outer Membrane Protein, OMPs) 분석Experimental Example 5. Outer Membrane Protein (OMPs) Analysis
박테리아 OMP를 분리하여 이를 분석하였다. 구체적으로, LB broth에서 배양된 박테리아 세포를 2,500 xg 에서 20분 동안 원심분리하여 수확했다. 박테리아 세포 펠렛을 10 mM N-2-hydroxyethylpiperazine N′-2-ethanesulfonic acid에 현탁시키고, Vibra-cell 초음파 액체 처리기(Sonics & Materials Inc., Newtown, CT, USA)로 초음파 처리했다. 세포 용해물의 박테리아 막 분획은 100,000 xg에서 45 Ti 회전자(Beckman Coulter, Brea, CA, USA)를 사용하여 초원심분리에 의해 수집되었다. 4°C에서 1시간 동안 초원심분리 후, 막 분획을 1% N-lauroylsarcosine solution(Sarkosyl, Sigma-Aldrich)을 함유하는 완충액에 재현탁하여 내막 단백질을 가용화하고 제거하였다. 사르코실 처리된 OMP 분획을 위에서 설명한 대로 초원심분리를 사용하여 정제하고 마지막으로 인산완충식염수에 재현탁했다. OMP의 농도는 Pierce BCA 단백질 분석 키트(Thermo Scientific Inc.)를 사용하여 측정하였다. 10 μg의 OMP를 12% SDS-PAGE를 사용하여 분석하고 Coomassie Brilliant Blue로 염색하여 시각화했다. Bacterial OMPs were isolated and analyzed. Specifically, bacterial cells cultured in LB broth were harvested by centrifugation at 2,500 xg for 20 minutes. Bacterial cell pellets were suspended in 10 mM N-2-hydroxyethylpiperazine N′-2-ethanesulfonic acid and sonicated with a Vibra-cell ultrasonic liquid processor (Sonics & Materials Inc., Newtown, CT, USA). The bacterial membrane fraction of cell lysates was collected by ultracentrifugation using a 45 Ti rotor (Beckman Coulter, Brea, CA, USA) at 100,000 xg. After ultracentrifugation at 4 °C for 1 hour, the membrane fraction was resuspended in a buffer containing 1% N-lauroylsarcosine solution (Sarkosyl, Sigma-Aldrich) to solubilize and remove inner membrane proteins. The sarcosyl-treated OMP fraction was purified using ultracentrifugation as described above and finally resuspended in phosphate buffered saline. The concentration of OMP was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific Inc.). 10 μg of OMP was resolved using 12% SDS-PAGE and visualized by staining with Coomassie Brilliant Blue.
관심있는 단백질 밴드는 펩타이드 질량 핑거프린팅 방법으로 분석하였다. 간단히 말해서, SDS-PAGE 젤의 단백질을 추출한 후 트립신(Promega)으로 소화시켰다. 소화된 펩타이드; 및 50% acetonitrile/0.1% trifluoroacetic acid(Sigma-Aldrich) 중 α-cyano-4-hydroxycinnamic acid과 혼합하고 펩타이드 혼합물을 matrix-assisted laser desorption ionization-time-of-flight mass spectrometry(Microflex LRF 20, Bruker Daltonics, MA, USA)를 사용하여 분석했다. 질량 스펙트럼은 600-3,000의 m/z 범위에서 수집되었으며 trypsin autodigestion peak(m/z 842.510, 2211.1046)에 의해 내부적으로 보정되었다. MASCOT 서버 2.3 버전(Matrix Science, 런던, 영국)을 사용하여 질량 스펙트럼을 분석했다. MASCOT 점수가 >73인 단백질은 표적 단백질과 유의하게 일치하는 것으로 간주되었다(P < 0.05).Protein bands of interest were analyzed by peptide mass fingerprinting method. Briefly, proteins were extracted from SDS-PAGE gels and then digested with trypsin (Promega). digested peptide; and α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid (Sigma-Aldrich), and the peptide mixture was subjected to matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Microflex LRF 20, Bruker Daltonics , MA, USA). Mass spectra were collected in the m/z range of 600-3,000 and internally calibrated by the trypsin autodigestion peak (m/z 842.510, 2211.1046). Mass spectra were analyzed using the MASCOT server version 2.3 (Matrix Science, London, UK). Proteins with a MASCOT score >73 were considered significantly matched to the target protein (P < 0.05).
실험예 6. RNA 추출 및 정량적 실시간 역전사(qRT-PCR)Experimental Example 6. RNA extraction and quantitative real-time reverse transcription (qRT-PCR)
RNA 분해를 방지하기 위해 LB broth에서 배양된 박테리아 세포 또는 살모넬라에 감염된 동물 세포를 제조사의 지침에 따라 RNA protect Bacteria Reagent(Qiagen, Hilden, Germany) 또는 RNAlater 안정화 용액(Ambion, Austin, TX, USA)으로 처리하였다. RNeasy mini kit(Qiagen)를 사용하여 박테리아의 총 RNA를 분리하고 TURBO DNA-free 키트(Ambion)를 사용하여 잔류 염색체 DNA를 제거했다. RNA to cDNA EcoDry Premix with random hexamers(Clontech Laboratory, CA, USA)를 사용하여 RNA를 cDNA로 역전사하였고, 10 ng의 input RNA에 해당하는 cDNA를 각 qPCR에서 주형으로 사용했다. Primer는 Primer Express Software 3.0 버전(Applied Biosystems, MA, USA)을 사용하여 설계하였고, 설계된 프라이머 서열은 상기 표 2와 같다.To prevent RNA degradation, bacterial cells cultured in LB broth or Salmonella-infected animal cells were incubated with RNA protect Bacteria Reagent (Qiagen, Hilden, Germany) or RNAlater stabilizing solution (Ambion, Austin, TX, USA) according to the manufacturer's instructions. processed. Bacterial total RNA was isolated using the RNeasy mini kit (Qiagen) and residual chromosomal DNA was removed using the TURBO DNA-free kit (Ambion). RNA was reverse transcribed into cDNA using RNA to cDNA EcoDry Premix with random hexamers (Clontech Laboratory, CA, USA), and cDNA corresponding to 10 ng of input RNA was used as a template in each qPCR. Primers were designed using Primer Express Software version 3.0 (Applied Biosystems, MA, USA), and the designed primer sequences are shown in Table 2 above.
증폭된 PCR 산물을 검출하기 위해 SYBR 녹색 시약(Power SYBR Green PCR Master Mix, Applied Biosystems)과 함께 StepOnePlus 실시간 PCR 기기(Applied Biosystems)를 사용하여 qPCR을 수행했다. 각 유전자의 상대적 발현 수준은 gyrB의 수준으로 정규화되었으며, 독립적으로 추출된 RNA 샘플을 사용하여 세 가지 테스트의 평균으로 표시하였다.qPCR was performed using a StepOnePlus real-time PCR instrument (Applied Biosystems) with SYBR Green reagent (Power SYBR Green PCR Master Mix, Applied Biosystems) to detect amplified PCR products. The relative expression level of each gene was normalized to that of gyrB and expressed as the average of three tests using independently extracted RNA samples.
실험예 7. 침습(invasion) 및 생존(survival) 분석Experimental Example 7. Invasion and survival analysis
HeLa 인간 상피 세포 및 RAW264.7 쥐 대식세포를 10% FBS(fetal bovine serum)이 보충된 Dulbecco의 변형된 Eagle 배지(DMEM)에서 성장시키고, 1×105 및 2×105 세포/웰의 밀도로 24웰 배양 플레이트에 각각 접종했다. 밤새도록 LB broth에서 배양한 살모넬라 세포를 감염 다중도(multiplicity of infection) 100으로 동물 세포에 첨가하였다. 박테리아 감염 30분 후 세포를 PBS로 3회 세척하고, 젠타마이신(100 μg/mL)을 함유하는 새로운 DMEM으로 1.5시간 동안 보충했다. 세포외 박테리아를 제거하기 위해 나머지 감염 기간 동안 배지를 10μg/mL 젠타마이신을 함유하는 새로운 DMEM으로 교체하였다. HeLa human epithelial cells and RAW264.7 murine macrophages were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS (fetal bovine serum), at densities of 1×10 5 and 2×10 5 cells/well. were inoculated into 24-well culture plates, respectively. Salmonella cells cultured in LB broth overnight were added to animal cells at a multiplicity of infection of 100. After 30 min of bacterial infection, cells were washed 3 times with PBS and supplemented with fresh DMEM containing gentamicin (100 μg/mL) for 1.5 h. The medium was replaced with fresh DMEM containing 10 μg/mL gentamicin for the remainder of the infection period to remove extracellular bacteria.
침습 분석을 위해 감염된 HeLa 세포를 감염 후 2시간에 1% Triton X-100을 사용하여 용해시켰고, 세포 용해물을 희석하고 LB 한천에 플레이팅했다. 생존 분석에서 감염된 RAW264.7 세포는 감염 후 10시간에 용해시켰고, 세포 내 박테리아는 침입 분석에 대해 설명된 것과 동일한 방식으로 계수하였다.For the invasion assay, infected HeLa cells were lysed using 1% Triton X-100 2 hours post infection, cell lysates were diluted and plated on LB agar. RAW264.7 cells infected in the survival assay were lysed 10 hours after infection, and intracellular bacteria were counted in the same way as described for the invasion assay.
실험예 8. 동물 윤리Experimental Example 8. Animal Ethics
모든 동물 실험은 국제법 및 정책 지침(실험동물 보호 및 이용에 관한 지침, 국립 아카데미 출판부, 8판)에 따라 수행되었으며, 강원대학교 동물연구소 동물관리위원회의 승인을 받았다. 승인번호 KW-160201-1).All animal experiments were conducted in accordance with international laws and policy guidelines (Guidelines for the Protection and Use of Laboratory Animals, National Academy Press, 8th Edition) and were approved by the Animal Care Committee of the Institute of Animal Research, Kangwon National University. Approval No. KW-160201-1).
실험예 9. 마우스 감염 실험Experimental Example 9. Mouse Infection Experiment
마우스 감염 실험을 위하여, LD50(half-maximal lethal dose)를 계산하였다. 6주령 암컷 BALB/c 마우스(Orient Bio Inc., Seongnam, Korea)에 102 내지 107 CFU/mouse로 연속 희석된 박테리아 세포를 복강 내 주사하였다. 감염 후 2주 동안 마우스를 모니터링하고 죽은 마우스의 수를 기록하여 LD50 값을 추정하였다. 아프고 빈사 징후가 있는 마우스는 인도적 차원에서 안락사시켰다. LD50 값은 다음의 계산식으로 계산하였다.For mouse infection experiments, LD 50 (half-maximal lethal dose) was calculated. 6-week-old female BALB/c mice (Orient Bio Inc., Seongnam, Korea) were intraperitoneally injected with serially diluted bacterial cells at 10 2 to 10 7 CFU/mouse. Mice were monitored for 2 weeks after infection and the number of dead mice was recorded to estimate LD 50 values. Sick and moribund mice were humanely euthanized. The LD 50 value was calculated by the following formula.
log10[50% end point]=A+(BXC)log10[50% end point]=A+(BXC)
* A = log 10[감염량 다음으로 사망률이 50% 미만인 감염선량]* A = log 10 [infectious dose at which the mortality rate is less than 50% following the infected dose]
* B = 로그의 차이 = [50% -(감염량에서 사망률 다음으로 50% 미만)]/[(다음으로 사망률이 50% 이상) -(다음으로 사망률이 50% 미만) ]* B = log difference = [50% - (next mortality rate less than 50% in infection dose)]/[(next mortality rate greater than 50%) - (next mortality rate less than 50%) ]
* C = log 10 [시험처리 연구에 사용된 연속 감염 용량 간의 차이]* C = log 10 [difference between consecutive infection doses used in challenge studies]
생체 내 독성 테스트는 104 CFU/dose에서 박테리아 세포로 복강 내 감염된 4마리의 BALB/c 마우스 그룹을 사용하여 수행하였다. 감염 후 14일(dpi)에 마우스를 희생시키고 신체 및 비장의 무게를 측정했다. 비장은 후속적으로 TissueLyser II(Qiagen, USA)를 사용하여 30Hz에서 1분 동안 균질화하였고, 용해물을 연속적으로 희석하였다. 박테리아 세포를 계수하기 위하여, 연속 희석액을 Salmonella-Shigella(SS) 한천(Difco, Becton Dickinson, MD, USA)에 플레이팅했다.In vivo toxicity testing was performed using groups of 4 BALB/c mice infected intraperitoneally with bacterial cells at 10 4 CFU/dose. Mice were sacrificed on day 14 post infection (dpi) and body and spleen weights were measured. Spleens were subsequently homogenized using a TissueLyser II (Qiagen, USA) at 30 Hz for 1 minute, and lysates were serially diluted. To enumerate bacterial cells, serial dilutions were plated on Salmonella-Shigella (SS) agar (Difco, Becton Dickinson, MD, USA).
실험예 10. 마우스 예방 접종 및 도전 실험Experimental Example 10. Mouse Vaccination and Challenge Experiment
암컷 BALB/c 마우스를 각각 6마리의 마우스 그룹으로 나누고 각각 103 CFU/dose 및 104 CFU/dose로 복강 내 주사하여 야생형 및 ΔyjeK 돌연변이 균주로 면역화시켰다. 음성 대조군은(N control)은 균주 대신 PBS를 접종하였다. 마우스 면역 후 0, 7, 14, 28 및 35일에 혈액을 수집하고 혈청을 사용할 때까지 -20°C에서 보관하였다. 28일 후, 마우스에 ST2173 균주 108 CFU/dose(보호 분석) 또는 1010 CFU/dose(생존 분석)를 경구 투여하였다. 양성 대조군(P control)은 PBS를 투여한 다음 동일한 용량의 ST2173으로 챌린지했다.Female BALB/c mice were divided into groups of 6 mice each and immunized with wild-type and ΔyjeK mutant strains by intraperitoneal injection with 10 3 CFU/dose and 10 4 CFU/dose, respectively. Negative control (N control) was inoculated with PBS instead of the strain. Blood was collected at 0, 7, 14, 28 and 35 days after mouse immunization and serum was stored at -20 °C until use. After 28 days, mice were orally administered with 10 8 CFU/dose (protection assay) or 10 10 CFU/dose (survival assay) of the ST2173 strain. A positive control (P control) was challenged with the same dose of ST2173 after administration of PBS.
마우스 보호 분석에서, 항원-공격된 마우스는 면역화 후 35일에 희생시켰다. 희생된 마우스로부터 간 및 비장을 포함한 장기를 수집하였고, SS 한천에 도말하였다. 마우스 생존 분석에서, 항원-공격된 마우스를 추가로 24일 동안 모니터링하고 사망률을 기록했다.In the mouse protection assay, antigen-challenged mice were sacrificed 35 days after immunization. Organs including liver and spleen were collected from sacrificed mice, and plated on SS agar. In mouse survival assays, challenged mice were monitored for an additional 24 days and mortality was recorded.
실험예 11. ELISA(Enzyme-Linked Immunosorbent Assay)Experimental Example 11. ELISA (Enzyme-Linked Immunosorbent Assay)
혈청 내 특정 IgG 및 IgM 항체 반응은 이전에 설명한 대로 ELISA를 사용하여 측정되었다. 혈청 및 효소 접합체의 최적 농도는 면역화된 마우스 및 면역화되지 않은 마우스의 3중 혈청을 사용 하는 체커보드 테스트를 통해 결정하였다. 혈청과 효소 접합체 간의 최상의 결합 비율을 선택하여 S. Typhimurium에 특이적인 IgG, IgM 및 IgG 서브클래스(IgG1 및 IgG2a) 항체 반응을 측정했다. 야생형 S. Typhimurium로부터 제조된 OMP을 마이크로플레이트(Thermo Scientific Inc.)에 0.5 μg/well로 첨가하고 4℃에서 밤새도록 배양하였다. 면역화된 마우스의 혈청을 1:200으로 희석하고 코팅된 웰에 첨가하였다. 1시간 배양 후, 마이크로플레이트를 Tween 20을 함유하는 PBS(TBST)로 세척하고, horseradish peroxidase-labeled goat anti-mouse IgG(1:30,000 희석; Bethyl Laboratories, TX, USA), anti-mouse IgM(1 :35,000 희석, Bethyl Laboratories), anti-mouse IgG1(1:25,000 희석, Bethyl Laboratories) 또는 anti-mouse IgG2a(1:30,000 희석, Bethyl Laboratories) 2차 항체를 처리하였다. 결합되지 않은 2차 항체를 0.05% PBST로 3회 세척하였다. 그 후 3,3’,5,5’-tetramethylbenzidine(Surmodics Inc., MN, USA) 기질을 각 웰에 첨가하고, 5분 동안 배양했다. 효소 반응은 H2SO4(0.5M) 100 ml를 첨가하여 정지시켰다. 항체 수준을 정량하기 위하여, Epoch 플레이트 판독기(BioTek, GA, USA)로 450 nm에서 흡광도를 측정하였다.Specific IgG and IgM antibody responses in serum were measured using ELISA as previously described. Optimal concentrations of serum and enzyme conjugate were determined by a checkerboard test using triplicate sera from immunized and non-immunized mice. S. by selecting the best binding ratio between serum and enzyme conjugates. Typhimurium-specific IgG, IgM and IgG subclass (IgG1 and IgG2a) antibody responses were measured. wild type S. OMP prepared from Typhimurium was added to a microplate (Thermo Scientific Inc.) at 0.5 μg/well and incubated overnight at 4°C. Serum from immunized mice was diluted 1:200 and added to the coated wells. After 1 hour of incubation, the microplate was washed with PBS (TBST) containing Tween 20, and horseradish peroxidase-labeled goat anti-mouse IgG (1:30,000 dilution; Bethyl Laboratories, TX, USA), anti-mouse IgM (1 :35,000 dilution, Bethyl Laboratories), anti-mouse IgG1 (1:25,000 dilution, Bethyl Laboratories) or anti-mouse IgG2a (1:30,000 dilution, Bethyl Laboratories) secondary antibody was treated. Unbound secondary antibody was washed 3 times with 0.05% PBST. Then, 3,3',5,5'-tetramethylbenzidine (Surmodics Inc., MN, USA) substrate was added to each well and incubated for 5 minutes. The enzymatic reaction was stopped by adding 100 ml of H 2 SO 4 (0.5 M). To quantify antibody levels, absorbance was measured at 450 nm with an Epoch plate reader (BioTek, GA, USA).
사이토카인 측정을 위하여, 비장 세포를 비장에서 분리한 후 계수하였다. RPMI-1640(GenDEPOT, TX, USA), 10mM HEPES(Duchefa) 및 10% FBS로 구성된 완전 배지에서 2X105 cells/well로 플레이팅했다. 플레이팅된 세포에 LPS(5 μg/mL, Sigma-Aldrich) 또는 가열로 인해 사멸된 S. Typhimurium 세포(HKC; 108 CFU/0.1 mL)를 처리하였고, 37°C, 5% CO2 조건에서 48시간 동안 배양하였다. 배양된 세포의 상등액을 수집하여, 사이토카인을 측정하였다. 상기 상등액은 사이토카인 측정 시까지 -20℃에서 보관하였다.For cytokine measurement, spleen cells were isolated from the spleen and counted. 2X10 5 cells/well were plated in complete medium consisting of RPMI-1640 (GenDEPOT, TX, USA), 10 mM HEPES (Duchefa) and 10% FBS. The plated cells were killed by LPS (5 μg/mL, Sigma-Aldrich) or heat. Typhimurium cells (HKC; 10 8 CFU/0.1 mL) were treated and cultured for 48 hours at 37°C and 5% CO 2 conditions. The supernatant of the cultured cells was collected and cytokines were measured. The supernatant was stored at -20 °C until cytokine measurement.
IFN-γ, IL-6 및 TNF-α의 농도는 제조사의 지침에 따라 사이토카인 ELISA 키트(MAX™ Standard, BioLegend, Inc., CA, USA)를 사용하여 결정하였다. 사이토카인의 농도는 Four Parameter Logistic Curve 소프트웨어(MyAssays Ltd., https://www.myassays.com/ )를 사용하여 계산하였다.Concentrations of IFN-γ, IL-6 and TNF-α were determined using a cytokine ELISA kit (MAX™ Standard, BioLegend, Inc., CA, USA) according to the manufacturer's instructions. Cytokine concentrations were calculated using Four Parameter Logistic Curve software (MyAssays Ltd., https://www.myassays.com/).
실험예 12. 통계 분석Experimental Example 12. Statistical Analysis
모든 값은 평균 ± 표준 편차로 표시하였다. P 값은 Tukey’s multiple comparison test로 분산의 일원 또는 이원 분석을 사용하여 계산되었다. P < 0.05는 통계적으로 유의한 것으로 간주하였다. 모든 통계 테스트는 GraphPad Prism 5(GraphPad Software Inc., CA, USA)로 수행하였다.All values are expressed as mean ± standard deviation. P values were calculated using one-way or two-way analysis of variance with Tukey's multiple comparison test. P < 0.05 was considered statistically significant. All statistical tests were performed with GraphPad Prism 5 (GraphPad Software Inc., CA, USA).
[실시예][Example]
실시예 1. ΔyjeK 돌연변이 균주의 생리학적 변화 분석Example 1. Analysis of physiological changes in ΔyjeK mutant strains
yjeK(1,029 bp) 및 efp(567 bp)의 인접한 두 유전자는 시작 코돈 사이에 40bp의 거리를 두고 서로 반대 방향으로 전사된다. 야생형 균주 ST1120의 yjek 유전자는 서열번호 2의 염기서열로 표시되며, λ red 재조합 방법을 사용하여 상기 야생형 yjek 유전자에서 서열번호 1의 뉴클레오티드를 결실시켰다. 즉, Δ yjeK 돌연변이 균주는 서열번호 1의 뉴틀레오티드가 결실된 yjek 유전자를 포함한다. efp 유전자는 결실 부위 근처에 있기 때문에 efp의 발현을 야생형과 결실 균주 사이에서 비교하였다. efp의 전사는 1yjeK 돌연변이 균주에서 결실에 의해 거의 영향을 받지 않아 Δ yjeK 돌연변이 균주에서 EF-P 활성에 대한 극성 효과의 가능성을 배제했다. Two adjacent genes, yjeK (1,029 bp) and efp (567 bp), are transcribed in opposite directions with a distance of 40 bp between start codons. The yjek gene of the wild-type strain ST1120 is represented by the nucleotide sequence of SEQ ID NO: 2, and the nucleotide sequence of SEQ ID NO: 1 was deleted from the wild-type yjek gene using the λ red recombination method. That is, the ΔyjeK mutant strain includes the yjek gene in which the nucleotide of SEQ ID NO: 1 is deleted. Since the efp gene is located near the deletion site, the expression of efp was compared between the wild-type and deletion strains. Transcription of efp was hardly affected by the deletion in the 1yjeK mutant strain, ruling out the possibility of a polar effect on EF-P activity in the ΔyjeK mutant strain.
Δ yjeK 돌연변이 균주의 성장 및 운동성을 분석한 결과는 각각 도 1A 및 B에 나타내었다.The results of analyzing the growth and motility of the ΔyjeK mutant strain are shown in FIGS. 1A and B, respectively.
도 1A 및 B에 나타낸 바와 같이, Δ yjeK 돌연변이 균주는 야생형 균주에 비해 성장 및 운동성이 현저히 감소한 것을 확인하였다.As shown in FIGS. 1A and B, it was confirmed that the growth and motility of the ΔyjeK mutant strain was significantly reduced compared to that of the wild-type strain.
또한, 분석 프로필 지수(analytical profile index, API) 테스트 결과, Δ yjeK 돌연변이 균주는 아르기닌 디하이드롤라제(arginine dihydrolase) 및 라이신 디카르복실라제와 관련된 생화학적 활성을 차단하여 돌연변이 균주에서 아르기닌 및 라이신 디카르복실화의 변경된 대사 경로를 나타내는 것을 확인하였다.In addition, as a result of the analytical profile index (API) test, the Δ yjeK mutant strain blocked the biochemical activities related to arginine dihydrolase and lysine decarboxylase, thereby reducing arginine and lysine decapsulants in the mutant strain. It was confirmed that it represents an altered metabolic pathway of reboxylation.
LPS 및 OMP 프로파일 분석 결과는 도 1C에 나타내었다.The LPS and OMP profile analysis results are shown in Figure 1C.
도 1C에 나타낸 바와 같이, Δ yjeK 돌연변이 균주가 긴 O-항원 부분에서 증가된 풍부함을 나타내고 다중 단백질의 수준을 변화시키는 것을 확인하였다. 또한 야생형 및 ΔyjeK 돌연변이 균주 사이의 차등 생산을 나타내는 6개의 OMP가 질량 분석법에 의해 확인되었다(도 1C의 화살표). 상기 OMP 6개에 대한 정보는 표 3에 나타내었다.As shown in Fig. 1C, it was confirmed that the ΔyjeK mutant strain showed increased abundance in the long O-antigen region and altered levels of multiple proteins. In addition, 6 OMPs showing differential production between wild-type and ΔyjeK mutant strains were identified by mass spectrometry (arrows in Fig. 1C). Information on the six OMPs is shown in Table 3.
| SpotsSpots | Identified proteinsIdentified proteins | Gene symbolsGene symbols | Protein sequence coverage [%]Protein sequence coverage [%] |
| aa | flagellin FliCflagellin FliC | fliCfliC | 7373 |
| bb | porin OmpDporin OmpD | ompDompD | 8080 |
| cc | OmpAOmpA | ompAompA | 4747 |
| dd | flagellin FljBflagellin FljB | fljBfljB | 4747 |
| ee | flagellin FliCflagellin FliC | fliCfliC | 4242 |
| ff | porin OmpDporin OmpD | ompDompD | 6969 |
한천 플레이트에서 Δ yjeK 균주의 운동성 감소에 따라 Δ yjeK 돌연변이 균주에서 flagellin FliC flagellin FliC의 발현이 야생형 균주보다 감소되고, porin OmpD의 발현이 야생형 균주보다 증가되었다. 또한, 가장 풍부한 OMP 중 두 가지인 OmpA와 OmpD는 Δ yjeK 균주에서 발현의 변화를 보일 것으로 예측되었다.As the motility of the ΔyjeK strain decreased on agar plates, the expression of flagellin FliC and flagellin FliC in the ΔyjeK mutant strain was decreased compared to the wild-type strain, and the expression of porin OmpD was increased compared to the wild-type strain. In addition, two of the most abundant OMPs, OmpA and OmpD, were predicted to show changes in expression in the ΔyjeK strain.
상기 OmpA는 살모넬라 감염에서 강력한 면역원성을 유도하고, 포린 OmpD는 덜 생성되어 산화 분자에 대한 박테리아 투과성을 감소시키고 숙주에서 박테리아 생존을 촉진한다. 포린과 유출 펌프를 형성하는 박테리아 OMP는 투과성을 감소시키거나(포린) 약물 수출을 증가(유출 펌프)함으로써 항생제에 대한 박테리아 내성에 영향을 준다. 따라서 Δ yjeK 돌연변이 균주에서 porin OmpD의 증가가 관찰된바, 항생제에 대한 내성을 추가로 확인하였다. 항생제에 대한 내성을 확인한 결과는 도 1D에 나타내었다.The OmpA induces strong immunogenicity in Salmonella infection, and less porin OmpD is produced, reducing bacterial permeability to oxidizing molecules and promoting bacterial survival in the host. Bacterial OMPs that form porins and efflux pumps affect bacterial resistance to antibiotics by reducing permeability (porins) or increasing drug export (efflux pumps). Therefore, as an increase in porin OmpD was observed in the ΔyjeK mutant strain, resistance to antibiotics was further confirmed. The results of confirming resistance to antibiotics are shown in FIG. 1D.
도 1D에 나타낸 바와 같이, Δ yjeK 돌연변이 균주는 NA, SXT 및 TE를 제외한 다른 항생제에 감수성이 있었다. 특히, Δ yjeK 돌연변이 균주 처리군에서 GM, N, K 및 AMC의 억제 영역은 각각 최대 77%(21.0 ± 0.0 mm), 45%(18.9 ± 0.5 mm), 36%(21.8 ± 1.1 mm), 34%(27.5 ± 0.7 mm)임을 확인하였다.As shown in Fig. 1D, the ΔyjeK mutant strain was susceptible to other antibiotics except NA, SXT and TE. In particular, in the ΔyjeK mutant strain treatment group, the inhibitory regions of GM, N, K, and AMC were up to 77% (21.0 ± 0.0 mm), 45% (18.9 ± 0.5 mm), 36% (21.8 ± 1.1 mm), and 34%, respectively. % (27.5 ± 0.7 mm).
상기 결과는 Δ yjeK 균주가 성장, 운동성, 대사 및 항생제 감수성에서 상당한 변화를 나타내었음을 의미한다.These results indicate that the ΔyjeK strain exhibited significant changes in growth, motility, metabolism, and antibiotic susceptibility.
실시예 2. ΔyjeK 돌연변이 균주의 약독화 특성 확인Example 2. Confirmation of attenuation characteristics of ΔyjeK mutant strain
박테리아가 상피 세포를 침범하고 대식세포 내부에서 생존하는 능력을 추가로 조사했으며 그 결과는 도 2A에 나타내었다.The ability of bacteria to invade epithelial cells and survive inside macrophages was further investigated and the results are shown in Figure 2A.
도 2A에 나타낸 바와 같이, Δ yjeK 돌연변이 균주는 감염 후 2시간에 야생형 박테리아보다 3.73배 빠른 속도로 숙주 상피세포에 들어갔으나, 감염 10시간 후 대식세포 내에서 완전히 증식하지 못하는 것을 확인하였다.As shown in FIG. 2A, the ΔyjeK mutant strain entered host epithelial cells 3.73 times faster than wild-type bacteria at 2 hours after infection, but failed to fully proliferate in macrophages 10 hours after infection.
야생형 및 Δ yjeK 돌연변이 균주 사이의 SPI-1 및 SPI-2 T3SS와 관련된 유전자 발현을 분석하였으며, 그 결과는 도 2B에 나타내었다.Gene expression related to SPI-1 and SPI-2 T3SS between wild-type and ΔyjeK mutant strains was analyzed, and the results are shown in FIG. 2B.
도 2B에 나타낸 바와 같이, 대식세포 내부에서 Δ yjeK 돌연변이 균주는 야생형 균주에 비해 SPI-1 수준이 증가되고, SPI-2 수준이 감소된 것을 확인하였다(도 2B, 0h). 이는 SPI-1/SPI-2 T3SS 관련 유전자의 발현이 변경되었음을 의미한다. 그러나 Δ yjeK 돌연변이 균주에서 이러한 차등 발현은 박테리아가 세포 내 환경에 적응하는 동안 완화되는 것을 확인하였다(도 2B, 10h).As shown in Figure 2B, it was confirmed that the level of SPI-1 was increased and the level of SPI-2 was decreased in the ΔyjeK mutant strain compared to the wild-type strain inside macrophages (Fig. 2B, 0h). This means that the expression of SPI-1/SPI-2 T3SS-related genes was altered. However, it was confirmed that this differential expression in the ΔyjeK mutant strain was alleviated while the bacteria adapted to the intracellular environment (Fig. 2B, 10h).
감염 마우스 모델을 사용하여 생체 내에서 Δ yjeK 돌연변이의 독성을 추가로 평가했으며, 그 결과는 표 4에 나타내었다.The toxicity of the ΔyjeK mutant was further evaluated in vivo using an infection mouse model, and the results are shown in Table 4.
표 4에 나타낸 바와 같이, ip 경로를 통해 BALB/c 마우스에 접종된 ΔyjeK 돌연변이 균주는 LD50 값이 106.5 CFU로, 야생형 ST1120(103.2 CFU)보다 약 100배 감소된 것을 확인하였다.As shown in Table 4, it was confirmed that the ΔyjeK mutant strain inoculated into BALB/c mice through the ip route had an LD 50 value of 10 6.5 CFU, about 100 times lower than wild-type ST1120 (10 3.2 CFU).
Δ yjeK 돌연변이 균주에 감염된 마우스를 희생시킨 후 비장 및 신체의 무게를 측정하였다. 또한 분리된 비장 내 박테리아 수를 계수하였다. 마우스 비장의 무게 및 비장 내 박테리아 수를 확인한 결과는 각각 도 3A 및 B에 나타내었다. 또한 희생된 마우스의 신체 무게를 측정한 결과는 도 3C에 나타내었다.After sacrifice of mice infected with the ΔyjeK mutant strain, spleen and body weights were measured. The number of bacteria in the isolated spleen was also counted. The results of confirming the weight of the mouse spleen and the number of bacteria in the spleen are shown in FIGS. 3A and B, respectively. In addition, the result of measuring the body weight of the sacrificed mouse is shown in Figure 3C.
도 3A에 나타낸 바와 같이, Δ yjeK 돌연변이 균주에 감염된 마우스의 임상 징후 및 박테리아 부담도 야생형 감염 그룹의 임상 징후와 명확하게 달랐다. Δ yjeK 돌연변이 균주 감염 후 비장의 무게가 유의하게 감소한 반면(Δ yjeK, 0.34 ± 0.05 g vs. wild-type, 0.72 ± 0.12 g), 야생형 균주 감염에서는 살모넬라 균주에 의한 비장종대(splenomegaly)가 관찰되었다.As shown in Fig. 3A, the clinical signs and bacterial burden of mice infected with the ΔyjeK mutant strain were also clearly different from those of the wild-type infected group. After infection with the ΔyjeK mutant strain, the weight of the spleen was significantly reduced (ΔyjeK, 0.34 ± 0.05 g vs. wild-type, 0.72 ± 0.12 g), whereas splenomegaly caused by the Salmonella strain was observed in the infection with the wild-type strain. .
도 3B에 나타낸 바와 같이, Δ yjeK 돌연변이 균주 처리군의 비장 내 박테리아 수는 4.6X104 CFU/g이며, 이는 야생형 균주 처리군(2.6X105 CFU/g)에 비해 현저히 낮은 것을 확인하였다.As shown in FIG. 3B, the number of bacteria in the spleen of the ΔyjeK mutant strain treatment group was 4.6X10 4 CFU/g, which was significantly lower than that of the wild-type strain treatment group (2.6X10 5 CFU/g).
도 3C에 나타낸 바와 같이, Δ yjeK 돌연변이 균주 및 야생형 균주 처리군은 모두 체중에 유의한 변화가 없는 것을 확인하였다.As shown in Figure 3C, it was confirmed that there was no significant change in the body weight of both the Δ yjeK mutant strain and the wild-type strain treatment group.
상기 결과는 Δ yjeK 돌연변이 균주에 감염된 마우스의 비장에서 관찰된 비장종대 및 박테리아 수 감소는 상기 균주가 생체 내에서 약독화되었음을 의미한다.The above results indicate that the spleen enlargement and decrease in the number of bacteria observed in the spleen of mice infected with the ΔyjeK mutant strain indicate that the strain is attenuated in vivo.
실시예 3. ΔyjeK 돌연변이 균주로 면역화된 마우스에서 면역 반응 분석Example 3. Analysis of immune responses in mice immunized with ΔyjeK mutant strains
3-1. 혈청 항체 분석3-1. Serum antibody assay
ΔyjeK 돌연변이체의 면역학적 효과는 BALB/c 마우스에서 감염 후 생성된 혈청 항체(IgG 및 IgM)의 수준을 측정하였다. IgG 및 IgM의 발현 수준을 분석한 결과는 각각 도 4A 및 B에 나타내었다.The immunological effect of the ΔyjeK mutant was measured by the level of serum antibodies (IgG and IgM) produced after infection in BALB/c mice. The results of analyzing the expression levels of IgG and IgM are shown in FIGS. 4A and B, respectively.
도 4A 및 B에 나타낸 바와 같이, IgG 및 IgM 항체의 수준은 14 dpi까지 야생형 및 Δ yjeK 돌연변이 균주로 감염된 그룹은 유사하였다. 28 dpi에서 Δ yjeK 돌연변이 균주에 의한 면역화로 인해 IgG 및 IgM 항체의 수준은 14 dpi에 비해 각각 1.7 및 1.5배 증가했다.As shown in Figures 4A and B, the levels of IgG and IgM antibodies were similar for groups infected with wild-type and ΔyjeK mutant strains up to 14 dpi. At 28 dpi, immunization with the ΔyjeK mutant strain resulted in a 1.7- and 1.5-fold increase in the levels of IgG and IgM antibodies compared to 14 dpi, respectively.
IgG2a/IgG1 비율은 미생물 감염에 대한 T 헬퍼(Th)1과 Th2 반응 사이의 면역 균형을 위한 대리 마커로 일반적으로 사용된다. 면역된 마우스의 IgG2a/IgG1 비율을 측정하였으며, 그 결과는 도 4C에 나타내었다.The IgG2a/IgG1 ratio is commonly used as a surrogate marker for the immune balance between T helper (Th)1 and Th2 responses to microbial infections. The IgG2a/IgG1 ratio of the immunized mice was measured, and the results are shown in FIG. 4C.
도 4C에 나타낸 바와 같이, ΔyjeK 돌연변이 균주로 면역된 마우스의 IgG2a/IgG1 비율은 2.32(28 dpi) 내지 3.66(14 dpi) 범위에 있고, 야생형 균주로 감염된 그룹의 IgG2a/IgG1 비율은 5.16(28 dpi) 내지 5.65(14 dpi)임을 확인하였다.As shown in Fig. 4C, the IgG2a/IgG1 ratio of the mice immunized with the ΔyjeK mutant strain ranged from 2.32 (28 dpi) to 3.66 (14 dpi), and the IgG2a/IgG1 ratio of the group infected with the wild-type strain was 5.16 (28 dpi). ) to 5.65 (14 dpi).
상기 결과는 야생형 및 ΔyjeK 돌연변이 균주 모두 BALB/c 마우스에서 Th2 매개 면역 반응보다 Th1 매개 면역 반응을 더 강력하게 자극했음을 의미한다. ΔyjeK 돌연변이 균주가 야생형 균주보다 더 높은 수준의 IgG를 유도하지만 더 낮은 IgG2a/IgG1 비율을 유도한다는 점을 고려할 때, ΔyjeK 돌연변이 균주를 이용한 면역화는 야생형 살모넬라균보다 더 강력하게 IgG2a 및 IgG1 관련 보호도 부여한다는 것을 의미한다.These results indicate that both the wild-type and ΔyjeK mutant strains stimulated the Th1-mediated immune response more strongly than the Th2-mediated immune response in BALB/c mice. Given that the ΔyjeK mutant strain induces higher levels of IgG than the wild-type strain but lower IgG2a/IgG1 ratio, immunization with the ΔyjeK mutant strain also conferred more potent IgG2a and IgG1-related protection than wild-type Salmonella means to do
3-2. 사이토카인 분석3-2. Cytokine analysis
면역 후 마우스의 비장을 28dpi로 채취하고 비장에서 분리한 비장세포를 S. Typhimurium LPS 및 HKC로 처리하여 살모넬라 면역원에 대한 사이토카인 생산 수준을 추정하였다. 면역화된 마우스 비장세포에서 사이토카인 IFN-γ, IL-6 및 TNF-α를 분석한 결과는 각각 도 5A 내지 C에 나타내었다.After immunization, the spleen of the mouse was collected at 28 dpi, and the splenocytes isolated from the spleen were treated with S. Typhimurium LPS and HKC to estimate the cytokine production level for the Salmonella immunogen. The results of analyzing the cytokines IFN-γ, IL-6 and TNF-α in the immunized mouse splenocytes are shown in FIGS. 5A to C, respectively.
도 5A 내지 C에 나타낸 바와 같이, 사이토카인 IFN-γ, IL-6 및 TNF-α는 면역화에 사용된 박테리아 균주에 관계없이 LPS 또는 HKC에 대한 반응으로 모두 증가했다. 흥미롭게도, Δ yjeK 돌연변이 균주를 사용한 면역은 야생형 살모넬라 에서 관찰된 것보다 LPS(2.24배) 및 HKC(1.5배)에 대한 IFN-γ 생산을 더 크게 촉진했다(도 5A). 그러나 전염증성 사이토카인 IL-6 및 TNF-α의 생성은 LPS 및 HKC 처리 둘 다에 대해 ΔyjeK 돌연변이 균주로 면역화한 후 더 낮은 정도로 유도되었다(도 5B 및 C).As shown in Figures 5A-C, the cytokines IFN-γ, IL-6 and TNF-α were all increased in response to LPS or HKC, regardless of the bacterial strain used for immunization. Interestingly, immunization with the ΔyjeK mutant strain promoted IFN-γ production to a greater extent for LPS (2.24-fold) and HKC (1.5-fold) than was observed for wild-type Salmonella (Fig. 5A). However, production of the pro-inflammatory cytokines IL-6 and TNF-α was induced to a lesser extent after immunization with the ΔyjeK mutant strain for both LPS and HKC treatment (FIGS. 5B and C).
상기 결과는 Δ yjeK 돌연변이 균주로 면역화된 마우스는 숙주 기관에서 전신 감염 징후 없이 IgG 및 IgM 항체를 유도하고, 동시에 살모넬라의 면역원성 항원에 대한 챌린지에 대해 사이토카인 생산을 조절한다는 것을 시사한다.These results suggest that mice immunized with the ΔyjeK mutant strain induce IgG and IgM antibodies without signs of systemic infection in the host organ, and at the same time modulate cytokine production in response to challenge with immunogenic antigens of Salmonella.
실시예 4. Δ yjeK 돌연변이 균주로 면역된 마우스에서 살모넬라증 예방 효과 확인Example 4. Confirmation of salmonellosis preventive effect in mice immunized with ΔyjeK mutant strain
도 6A와 같은 실험 일정으로, ΔyjeK 돌연변이 균주를 이용한 면역화에 의한 예방 효과를 BALB/c 마우스에서 평가하였다. 구체적으로, 후보 백신인 ΔyjeK 돌연변이 균주를 마우스에 복강 내 투여하여 면역화시켰다. 면역화된 마우스(28 dpi)에 야생형 S. Typhimurium 2173(ST2173) 균주를 경구 감염시켰다. 상기 야생형 S. Typhimurium 2173(ST2173) 균주가 감염된 마우스의 살모넬라 감염 예방 효과를 분석하였다.According to the experimental schedule shown in FIG. 6A, the preventive effect of immunization with the ΔyjeK mutant strain was evaluated in BALB/c mice. Specifically, the ΔyjeK mutant strain, which is a candidate vaccine, was immunized by intraperitoneal administration to mice. Immunized mice (28 dpi) were orally infected with wild-type S. Typhimurium 2173 (ST2173) strain. The effect of preventing Salmonella infection in mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain was analyzed.
4-1. 임상증상 관찰4-1. Observation of clinical symptoms
야생형 S. Typhimurium 2173(ST2173) 균주가 감염된 마우스의 임상증상을 관찰하였다. 그 결과, 사전 예방접종 없이 ST2173에 감염된 생쥐에서 거친 털, 딱딱하고 감긴 눈, 식욕 부진, 구부린 자세, 떨림, 체중 감소 및 사망을 포함한 살모넬라 감염의 전형적인 임상 증상이 관찰되었다.Clinical symptoms of mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain were observed. As a result, ST2173-infected mice without prior vaccination showed typical clinical signs of Salmonella infection, including coarse fur, hard, closed eyes, anorexia, hunched posture, tremors, weight loss and death.
4-2 체중 측정4-2 Weight measurement
상기 야생형 S. Typhimurium 2173(ST2173) 균주가 감염된 마우스의 체중을 측정하였으며, 그 결과는 도 6B에 나타내었다.The body weight of mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain was measured, and the results are shown in FIG. 6B.
도 6B에 나타낸 바와 같이, 35 dpi에서 야생형 S. Typhimurium 2173(ST2173) 균주가 감염된 마우스의 체중은 ST2173 감염에 의해 약 77%로 감소한 것을 확인하였다. 그러나 Δ yjeK 돌연변이 균주로 면역된 마우스는 ST2173 감염 후 7일 동안 체중 증가를 나타내어 살모넬라 균주를 주사하지 않은 건강한 마우스와 유사한 체중을 나타냈다.As shown in FIG. 6B, it was confirmed that at 35 dpi, the body weight of mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain was reduced by about 77% by ST2173 infection. However, mice immunized with the ΔyjeK mutant strain showed weight gain for 7 days after infection with ST2173, similar to healthy mice not injected with the Salmonella strain.
4-3. 간 및 비장의 박테리아 수 분석4-3. Analysis of the number of bacteria in the liver and spleen
상기 야생형 S. Typhimurium 2173(ST2173) 균주가 감염된 마우스를 희생시킨 후 간 및 비장의 무게를 측정하였고, 간 및 비장 내의 박테리아 수를 분석하였다. 간의 무게 및 박테리아 수를 분석한 결과는 도 6C에 나타내었고, 비장의 무게 및 박테리아 수를 분석한 결과는 도 6D에 나타내었다.After the mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain were sacrificed, the weight of the liver and spleen was measured, and the number of bacteria in the liver and spleen was analyzed. The results of analyzing the weight and number of bacteria in the liver are shown in FIG. 6C, and the results of analyzing the weight and number of bacteria in the spleen are shown in FIG. 6D.
도 6B 및 C에 나타낸 바와 같이, 후보 백신으로 면역된 마우스(Δ yjeK 돌연변이 균주 접종 그룹)에서는 간과 비장의 비대의 징후가 없었지만, 사전 예방접종을 하지 않은 공격받은 마우스는 간 및 비장이 비대했다. 또한 후보 백신으로 면역된 마우스(3.0 X 103 CFU/g liver 및 4.8 X 103 CFU/g spleen)의 간 및 비장의 세균 부담은 야생형 살모넬라로 면역된 마우스(2.3 X 104 CFU/g liver 및 7.5 × 104 CFU/g spleen) 및 면역되지 않은 마우스(positive challenge control: 3.9 X 105 CFU/g liver 및 2.5 X 106 CFU/g spleen)에 비해 유의하게 완화되었다.As shown in Fig. 6B and C, mice immunized with the candidate vaccine (ΔyjeK mutant strain inoculated group) showed no signs of liver and spleen enlargement, whereas challenged mice without prior vaccination had enlarged livers and spleens. In addition, the bacterial burden in the liver and spleen of mice immunized with the candidate vaccine (3.0 X 10 3 CFU/g liver and 4.8 X 10 3 CFU/g spleen) was higher than that of mice immunized with wild-type Salmonella (2.3 X 10 4 CFU/g liver and 7.5 × 10 4 CFU/g spleen) and unimmunized mice (positive challenge control: 3.9 X 10 5 CFU/g liver and 2.5 X 10 6 CFU/g spleen).
4-4. 생존 분석4-4. survival analysis
100 LD50의 박테리아 용량으로 ST2173의 경구 감염 후 면역화된 마우스의 생존율을 모니터링하여 후보 백신의 예방 효과를 평가하였다. 평가 결과는 도 6E에 나타내었다.The prophylactic effect of the candidate vaccine was evaluated by monitoring the survival of immunized mice after oral infection with ST2173 at a bacterial dose of 100 LD 50 . The evaluation results are shown in Fig. 6E.
도 6E에 나타낸 바와 같이, 살모넬라 균주로 면역화되지 않은 양성 대조군(P control)의 대부분은 ST2173 감염 후 1주 이내에 사망했다. 그러나 후보 백신으로 면역된 마우스(Δ yjeK)는 모니터링 기간 동안 ST2173 감염에서 살아남았다. 야생형 살모넬라균에 미리 감염된 마우스(WT)는 24dpi에서 독성 균주에 대한 공격에 대해 75% 생존했다.As shown in Fig. 6E, most of the positive control (P control) not immunized with the Salmonella strain died within 1 week after infection with ST2173. However, mice immunized with the candidate vaccine (ΔyjeK) survived ST2173 infection during the monitoring period. Mice pre-infected with wild-type Salmonella (WT) survived 75% challenge with the virulent strain at 24 dpi.
상기 결과는 후보 백신인 Δ yjeK 돌연변이 균주가 살모넬라균에 대한 완전한 보호를 제공할 수 있음을 의미한다.These results indicate that the candidate vaccine, the ΔyjeK mutant strain, can provide complete protection against Salmonella.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다.In the above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (10)
- 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움(Salmonella typhimurium) 균주.A Salmonella typhimurium strain containing a YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted.
- 제1항에 있어서,According to claim 1,상기 균주는 아르기닌 디하이드롤라제(arginine dihydrolase) 및 라이신 디카르복실라제(lysine decarboxylation)와 관련된 생화학적 활성을 차단하는 것인, 균주.The strain is to block the biochemical activity associated with arginine dihydrolase and lysine decarboxylase (lysine decarboxylation), the strain.
- 제1항에 있어서,According to claim 1,상기 균주는 AMC(amoxicillin/clavulanic acid), 암피실린(ampicillin, AM), 세팔로틴(cephalothin, CF), 겐타마이신(gentamicin, GM), 카나마이신(kanamycin, K), 네오마이신(neomycin, N) 및 SAM(ampicillin/sulbactam)으로 이루어진 군에서 선택된 1종 이상의 항생제에 대해 항생제 감수성을 나타내는 것인, 균주.The strain is amoxicillin/clavulanic acid (AMC), ampicillin (AM), cephalothin (CF), gentamicin (GM), kanamycin (K), neomycin (N) and SAM (ampicillin / sulbactam) that exhibits antibiotic sensitivity to one or more antibiotics selected from the group consisting of, the strain.
- 제1항에 있어서,According to claim 1,상기 균주는 flagellin FliC의 발현이 야생형 균주보다 감소되고, porin OmpD의 발현이 야생형 균주보다 증가된 것인, 균주.Wherein the strain has reduced expression of flagellin FliC compared to the wild-type strain and increased expression of porin OmpD compared to the wild-type strain.
- 제1항에 있어서,According to claim 1,상기 균주는 숙주 내에서 비장종대(splenomegaly)를 유도하지 않는 것인, 균주.Wherein the strain does not induce splenomegaly in the host.
- 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 포함하는 살모넬라증(Salmonellosis) 예방 또는 치료용 백신 조성물.A Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. At least one selected from the group consisting of A vaccine composition for preventing or treating Salmonellosis.
- 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 포함하는 살모넬라균에 대한 면역원성 조성물. A Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. At least one selected from the group consisting of An immunogenic composition against Salmonella.
- 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 포함하는 살모넬라증 예방 또는 개선용 사료 조성물.A Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. At least one selected from the group consisting of A feed composition for preventing or improving salmonellosis.
- 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 포함하는 살모넬라증 예방 또는 개선용 사료 첨가제 조성물.A Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. At least one selected from the group consisting of A feed additive composition for preventing or improving salmonellosis.
- 서열번호 1의 염기서열로 표시되는 뉴클레오티드가 결실된 YjeK 유전자를 포함하는 살모넬라 티피뮤리움 균주, 상기 균주의 배양물, 상기 배양물의 농축물 및 상기 배양물의 건조물로 이루어진 군에서 선택된 1종 이상을 이를 필요로 하는 개체에 투여하는 단계;를 포함하는 살모넬라증의 예방 또는 치료방법.At least one selected from the group consisting of a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dried product of the culture A method for preventing or treating salmonellosis comprising; administering to a subject in need thereof.
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