KR20140053475A - Rfah mutated salmonella typhimurium and a composition for food poisoning prevention using the same - Google Patents

Abstract

The present invention relates to a Salmonella typhimurium strain of which a part of a rfaH gene is deficient and a vaccine composition for preventing food poisoning using the same. The present invention provides an attenuated Salmonella typhimurium strain of which a part of the rfaH gene, which is a transcription regulator, is deleted, so that infectivity, intracellular survival rate, human serum resistance, and swarming motility of the Salmonella typhimurium strain are reduced and vulnerability to anti-microbial peptides is increased, and such Salmonella typhimurium strain can be effectively used as a vaccine for preventing and treating food poisoning of various livestock products.

Description

A rfaH-deficient Salmonella typhimurium strain and a food poisoning prevention vaccine composition using the strain,

The present invention relates to a Salmonella typhimurium strain in which a part of the rfaH gene is deficient and a vaccine composition for preventing food poisoning using the strain.

Salmonella is morphologically or physiologically similar to E. coli but has been classified as an independent genus by K. Kauffmann et al. For medical convenience. Salmonella was found in Salmonella in 1885 by Salmon and Smith in a pig that died of Don cholera ( Salmonella choleraesuis ) have been isolated from patients with enteritis, gastroenteritis and various diseases. It has also been isolated from healthy animals such as cows, pigs, pigs, goats, dogs, cats, and the environment, including chickens. Salmonella spp. Has been reported to date to more than 2,500 serotypes, for example, S. Pullorum, S. gallinarum) and no host specificity of strains in S. choleraesuis, chickens in S. Typhi, cattle in human S. Dublin, pigs (S. Typhimurim, S. Enteriditis, S. Newprot, etc.), among which the serotypes causing human food poisoning through livestock products are the latter.

Salmonella is a pathogenic bacterium that causes the most number of patients with Shigella causing dysentery in Korea. It is a very dangerous bacteria that can be transmitted to the human body through circulation of aquatic animal contaminated with Salmonella. Salmonella is a gram negative bacillus that does not form spores and is found as parasites in various animals.

Salmonella infection occurs in several forms, but food poisoning and enteritis form is the most common symptom. In the past, the cause of food poisoning in Korea was mainly caused by Shigella. Recently, the incidence of food poisoning by Salmonella has been gradually increasing. In 2000, more than 2,500 cases of foodborne illnesses due to Salmonella were found to be significantly higher than other foodborne pathogens such as Staphylococcus aureus (about 1,000 cases) and Vibrio (about 200 cases) Of the total.

On the other hand, look at the distribution of serotypes of Salmonella food poisoning causes accounted for almost half of the food poisoning caused by Salmonella, the ratio of the S. Enteritidis accounted Salmonella serotypes isolated from people in Seoul from 1996 to 2005 and a 42% , Followed by S. Typhimurium (see Table 1).

 <Distribution of Salmonella serotypes isolated from food poisoning patients in Seoul area from 1996 to 2005> Serotype No, of Isolates Total
(%) 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 S. Enteritidis 12 10 51 135 58 32 60 10 7 41 416
(42.0) S. Typhimurium 7 7 18 42 10 3 8 12 One 7 115
(11.6) S. Typhi 10 15 73 51 19 50 10 4 14 10 256
(25.9) Other Salmonella 6 8 18 57 18 9 58 7 7 14 202
(20.4) Total 35 40 160 285 105 94 136 33 29 72 989

In addition, S. enteritidis is the highest and S. Typhimurium is the next most common species of salmonella serotype found in persons surveyed by the CDC.

On the other hand, the detection rate of salmonella serotype in livestock products varies depending on the species of the species. In Korea, the systematic distribution of serotype of Salmonella serotype has not yet been reported. In the Netherlands, S. Typhi, the cause of typhoid fever, was the most isolated in humans, but S. typhimurium or S. enteritidis has been most frequently isolated from the 1980s (Table 2). In addition, the pig can be separated most commonly S. Typhimurium, cattle in high host specificity and S. Typhimurium and S. Dublin separation Most S. Enteritidis is relatively low detection rate. In case of chicken, S. Enteritidis is the most isolated and S. typhimurium is the sixth. The interesting thing is the one who occupy the detection rate of Salmonella serotypes of S. Enteritidis levels showed the highest detection rate in chicken, beef and pork will that S. Typhimurium accounting for water level, These results show similar patterns across countries.

<Salmonella serotype most isolated in the Netherlands from 1984 to 2001> Source Serotype Data for isolates from indicated period 1996-2001 1990-1995 1984-1989 % (no.) Rank % (no.) Rank % (no.) Rank Human Typhimurium 32.0 (4, 470) 2 33.1 (5,666) 2 57.2 (16,049) One Enteritidis 43.6 (6,097) One 33.8 (6,642) One 5.4 (1,526) 2 Pig Typhimurium 66.4 (2,384) One 75.7 (2,378) One 66.0 (2,197) One Enteritis - > 10 - > 10 - > 10 Chicken Typhimurium 5.3 (336) 6 14.3 (2,669) 3 26.1 (5, 170) One Enteritidis 20.3 (1,290) One 15.0 (2,802) 2 5.5 (1,088) 6 Cattle Typhimurium 32.9 (702) 2 31.9 (1,670) 2 53.1 (1,706) One Enteritidis - > 10 - > 10 - > 10

Since 1988, the World Health Organization (WHO) has recommended that an effective live vaccine be inoculated into target animals to prevent the food poisoning of salmonella from animal products. In particular, S. Typhimurum is the most frequent and important Salmonella serotypes that cause food poisoning in humans due to the propagation of S. enteritidis and livestock products.

In order to ensure livestock safety, vaccine use is an indispensable factor in the prevention of salmonella in industrial animals rather than antibiotic treatment. Although the vaccine can be divided into inactivated vaccine and live bacterial vaccine, salmonella is generally protected by cell mediated immunity, so that live vaccine vaccine is being studied all over the world. In the case of inactivated vaccine, It is used as a reinforcement vaccination for high immunity induction after virulence vaccine because it can defend the penetration into the real organs. Therefore, S. Typhimurium is a preventive vaccine that must be developed in pigs, chickens, and cows that are used as livestock foods to reduce human food poisoning. It is a highly marketable vaccine product that can be used regardless of species, And the need for development for the promotion of public health is urgently needed.

1. Gonzalo Rojas, Soledad Saldias et al. 2001. The rfaH gene, which affects lipopolysaccharide synthesis in Salmonella enterica serovar Typhi, is differentially expressed during the bacterial growth phase. FEMS Microbiology Letters 204 123-128. 2. Javier Santander, Wei Xin, Zhao Yang, Roy Curtiss. 2010. The aspartate-semialdehyde dehydrogenase of Edwardsiella ictaluri and its use as balanced-lethal system in fish vaccinology. PLoS One. Dec 29, 2010 (12): e15944. 3. Kang HY, Dozois CM, Tinge SA, Lee TH, Curtiss R 3rd. 2002. Transduction-mediated transfer of unmarked deletion and point mutations through use of counterselectable suicide vectors. J Bacteriol. Jan; 184 (1): 307-12. 4. Gahring, L. C., F. Heffron, B. B. Finlay, and S. Falkow. 1990. Invasion and replication of Salmonella typhimurium in animal cells. Infect. Immun. 58: 443-448 5. Nagy G, Danino V, Dobrindti, Pallen M, Chaudhuri R, Eml, Hinton JC, and Hacker J. 2006. Down-regulation of key virulence factors makes Salmonella enterica serovar Typhimurium rfaH mutant a promising live-attenuated vaccine candidate . Infect Immun. 2006 Oct; 74 (10): 5914-25.

Accordingly, an object of the present invention is to provide a novel strain of Salmonella typhimurium.

Another object of the present invention is to provide a food poisoning prevention vaccine using the strain

It is still another object of the present invention to provide a method for producing the strain.

In order to achieve the object of the present invention, the present invention provides a Salmonella typhimurium strain having a mutant gene rfaH .

In one embodiment of the present invention, the nucleotide sequence of the mutated rfaH gene is SEQ ID NO: 1.

In one embodiment of the present invention, the Salmonella typhimurium strain is Salmonella enterica serovar Typhimurium having a phage type of U302.

In one embodiment of the present invention, the strain is attenuated.

In one embodiment of the present invention, the attenuation is characterized by an increase in susceptibility to antimicrobial peptides present in the cell.

In one embodiment of the present invention, the antimicrobial peptide is Polymyxin B.

In addition, the present invention provides a vaccine composition for preventing or treating food poisoning comprising Salmonella typhimurium strain and a pharmaceutically acceptable carrier.

Further, the present invention relates to a method for producing a recombinant vector comprising the steps of: (a) extracting genomic DNA from Salmonella typhimurium (phage type U302); (B) amplifying the rfaH gene from the genomic DNA; (C) cleaving a portion of the rfaH gene and inserting an antibiotic resistance marker; (D) inserting the rfaH partial deletion gene into the vector; (E) transforming said vector into E. coli; (F) joining the transformed Escherichia coli with Salmonella typhimurium strain; Provides rfaH mutant type Salmonella typhimurium strain manufacturing method comprising the; the Salmonella typhimurium step (g) selecting the rfaH mutant type strain of the strain.

In one embodiment of the present invention, the rfaH Characterized in that a polymerization chain reaction is used using the primers of SEQ ID NOS: 2 to 5 in order to delete a part of the gene.

In one embodiment of the present invention, the selection step is characterized by selecting Salmonella typhimurium strains that survive in LB medium and kill in M9 medium.

The present invention can be very useful as an attenuated vaccine for Salmonella typhimurium which is reported to cause food poisoning in various livestock by providing a novel Salmonella typhimurium strain lacking a part of the rfaH gene.

Figure 1 shows a schematic diagram for producing a vector lacking the rfaH gene.
Figure 2 is a DNA electrophoresis image showing the donor vector pMEG375 and the insert Frag 1 + KM + Frag 2.
FIG. 3 is rfaH This is a PCR result for identifying a mutant Salmonella typhimurium strain in which a gene is partially deleted.
4 is rfaH This is another PCR result for identifying a mutant Salmonella typhimurium strain with a partial deletion of the gene.
FIG. 5 is a sequencing result for identifying a mutant Salmonella typhimurium strain in which the rfaH gene is partially deleted.
6 (a) compares the growth curves (sequentially, OD value, CFU / ml, and CFU / ml log values) of the wild type strain and the rfaH mutant strain in the LB medium.
FIG. 6 (b) compares the growth curves (sequentially, OD value and log value of CFU / ml) of the wild type strain and the rfaH mutant strain grown on the M9 medium.
Fig. 7 (a) shows the expression of wild type and rfaH Lt; RTI ID = 0.0 &gt; of the &lt; / RTI &gt; mutant strains.
Fig. 7 (b) is a graph comparing infection rates of wild-type and rfaH mutant strains.

The present invention relates to a Salmonella typhimurium strain in which the gene rfaH has been mutated.

Gene expression is regulated by proteins that are transcription factors. All germs known to humans have a transcription factor called NusG, which regulates expression of almost all genes in bacteria. Therefore, without NusG, bacteria can not survive. On the other hand, in the course of evolution, the NusG gene has been specialized, resulting in the appearance of RfaH transcription factors. RfaH regulates only a few genes, unlike NusG. However, the expression of genes that determine the infectivity of bacteria such as E. coli is regulated by RfaH.

RfaH protein is a transcriptional regulator of Gram negative bacteria, and various operons that depend on RfaH have been identified. All genes affected by RfaH encode elements outside the cell membrane, and these factors are reported to be related to bacterial toxicity (infectivity). For example, capsules and LPS (lipopolysaccharide) protect bacteria from host defense mechanisms, hemin receptors provide essential infectious agents, and alpha-hemolysin The case acts as a cytotoxin.

The RfaH gene is inactivated and is known to be selectively activated when an appropriate target sequence of the bacterium is found. In other words, these genes play a role in causing pathogenic bacteria to infect host cells and, at the same time, protect the bacteria from the host immune response, and it is observed that RfaH activity is suppressed as much as possible, unless absolutely necessary.

As described above, the present inventors have designed the present invention to prepare a live Salmonella typhimurium live vaccine by mutating the transcriptional regulator RfaH gene, which is closely related to the host immune response upon infection of the host cell, as described above.

In order to mutate the RfaH gene, the present inventors deleted about 100 bp of the middle region of the RfaH gene and insert a kanamycin cassette in the center thereof, so that the function of RfaH is lost, but the kanamycin antibiotic can be used as a selection mark.

The RfaH gene for making the mutant form was obtained from the genomic DNA of Salmonella typhimurium (phage type U302). These genes were mutagenized by PCR, cloned into a pGEMT-T easy vector, and ligated into a donor vector, PMEG 375 The vector was cloned again and conjugated with Salmonella typhimurium.

The mutant strain Salmonella typhimurium produced as described above showed a remarkable difference in growth rate, infectivity and survival rate compared to the wild type strain. That is, the growth rate of the mutant type was remarkably decreased as compared with the wild type, and this tendency was more remarkable in the M9 medium than in the LB medium (FIG. 6). In addition, there was also a difference in the infectivity, and the wild type showed a much better infectivity than the mutant type, and the survival rate in the wild type was higher than that in the mutant type (FIG. 7).

In addition, the present inventors confirmed that the mutant strains significantly reduced resistance to human serum, swarming mobility, and resistance to an anti-microbial peptide (Table 3). Thus, the inventors of the present invention found that a part of the rfaH gene is mutated Type Salmonella typhimurium strain was attenuated, and the possibility of using the same as a vaccine for various diseases derived from Salmonella typhimurium strain could be predicted.

Strain Serum resistance (%) Swarming motility (cm) MIC of Polymyxin B
(μg / ml) ST-wild type 100.3 + - 0.48 4.97 + 0.15 4.8 ST- rfaH Del. 0 0.6 0.64

The nucleotide sequence showing the rfaH mutation in the mutant Salmonella typhimurium strain may be as shown in SEQ ID NO: 1. Also, the primers of SEQ ID NOS: 2 to 5 may be used to prepare the mutant gene of SEQ ID NO: 1. Of course, rfaH The mutant form may be modified and used in various ways in which its function is lost in addition to the above-mentioned SEQ ID NO: 1, and a primer for producing a mutant gene may be used by being modified in a manner other than the primers of SEQ ID NOS: 2 to 5 will be.

In the present invention, the strain is Salmonella typhimurium strain, and the phage type is U302, but other types of phage can be modified.

In the present invention, the mutant Salmonella typhimurium strain and a pharmaceutically acceptable carrier can be used as a vaccine composition for preventing food poisoning.

The host animal in which the vaccine composition of the present invention can cause an immune response may include poultry such as human, chicken, duck, pheasant, turkey, pig, goat, sheep, cattle, dog, cat and the like.

The vaccine of the present invention may be an attenuated live vaccine or a sadox vaccine, a subunit vaccine, a synthetic vaccine or a genetic engineering vaccine, but a live vaccine that induces an effective immune response desirable.

The term " live vaccine " as used herein means a vaccine comprising the active ingredient of a live bacterium. The term "attenuation" refers to artificially weakening the toxicity of living pathogens, mutating the genes involved in the essential metabolism of pathogens, inducing immune system stimulating only the immune system without causing disease in the body do. The attenuation of the bacteria can be achieved by ultraviolet (UV) irradiation, chemical treatment, or in vitro high-throughput subculture. Inactivation can also be accomplished by making a clear genetic change, for example by insertion of a particular deletion of the gene sequence known to provide toxicity or a sequence into the genome of the strain.

The term " Sadox vaccine "is also referred to as inactivated vaccine or killed vaccine, and is a vaccine containing dead bacteria.

The term "subunit vaccine" is a vaccine prepared by extracting only an antigen component capable of causing an immune function among the constituents of a germ. The vaccine can minimize side effects by inducing immune formation only at an antigenic site necessary for the defense of germs.

A "genetic engineering vaccine" may be a modification or removal of a specific gene that causes the pathogenicity of the bacteria.

In addition, the vaccine of the present invention may be mixed with inactivated cells or antigens used for the preparation of other vaccines used for preventing diseases caused by bacteria other than Salmonella occurring in host animals, Can be used as a mixed or complex vaccine that can prevent diseases together. The term "combined vaccine " refers to a vaccine in which different antiviral drugs are used together.

The vaccine composition of the present invention may also comprise naked nucleic acid compositions. In one embodiment of the invention, the nucleic acid may comprise a nucleotide sequence encoding the mutant RfaH protein of the invention. Methods of nucleic acid vaccination can be carried out by methods known in the art. For example, in U.S. Patent Nos. 6,063,385 and 6,472,375. The nucleic acid may be in the form of a plasmid or gene expression cassette. In one embodiment, the nucleic acid is provided surrounded by liposomes administered to the animal.

In addition, the vaccine composition of the present invention may further comprise at least one member selected from the group consisting of a solvent, an adjuvant, and an excipient. Examples of the solvent include physiological saline or distilled water. Examples of the immunity enhancer include Freund's incomplete or complete adjuvant, aluminum hydroxide gel, vegetable and mineral oil, etc. Examples of excipients include aluminum phosphate, aluminum hydroxide Side or aluminum potassium sulfate, but are not limited thereto and may further comprise materials used in the manufacture of vaccines well known to those skilled in the art.

The vaccine composition of the present invention may be prepared in oral or parenteral formulations, preferably in the form of parenteral injectable solutions and may be formulated in intradermal, intramuscular, intraperitoneal, intravenous, intranasal, or eidural routes &Lt; / RTI &gt;

The term "prophylactic" in the present invention means any action that inhibits or slows the onset of Salmonella infection by administration of the composition. The term "treatment" in the present invention means any action that improves or alleviates symptoms due to Salmonella infection by administration of the composition.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dose level will depend on the species and severity of the subject, age, sex, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration, the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

< Example  1>

Salmonella Tiffy myrium rfaH Mutant type  Produce

1-1. Used strains and plasmids

As a strain to mutate the rfaH gene, Salmonella Escherichia coli DH5a (Real Biotech Co., Ltd. ) was used as a strain to clone a conjugated portion ( rfaH 100bp deletion fragment) for mutagenizing rfaH , and typhimurium (ST15, phage type: U302) Escherichia coli X7213 (ATCC, USA) was used as a donor strain to be bound to Salmonella typhimurium (ST15, phage type: U302). The vector used to construct a deletion fragment lacking 100 bp of rfaH was pGEMT-T easy vector (Progma, Maison, USA), and the vector used for splicing was PMEG 375 (Progma, Maison, USA).

1-2. badge

Bacteria were cultured in LB at 37 ° C and 230 rpm with low salt LB (Bacto-trypton 10 g / L, Bacto-yeast extract 5 g / L, NaCl 5 g / L) The medium for the recombination confirmation of the rfaH gene was M9 minimal media (12.8 g / L NaH ₂ HPO ₄ 7H ₂ O, 3 g / L KH ₂ PO ₄ , 0.5 g / L NaCl, 1 g / L NH ₄ Cl, 20% / v) glucose was used.

1-3. rfaH Gene defect Cloning

For the experimental method, the method described in the preceding documents 2 and 3 was referred to. Salmonella typhimurium (ST15, phage type: U302) from the G-spinTM Genomic DNA Extraction Kit after using (INTRON, Sungnam, Korea) extracting the chromosomal DNA in accordance with the manufacturer's method, using a PCR (polymerase chain reaction) rfaH The gene was amplified from the chromosomal DNA of Salmonella typhimurium. In this case, a kanamycin cassette, a selection marker, was added to confirm the presence of the junction, and a primer added with restriction enzyme sites was prepared in Cosmo Genetech Co. (seoul, Korea). The primers are shown in Table 4 below, and the PCR conditions are shown in Table 5 below.

<Primer used in PCR> Primer Name Sequence F1 AAA GAG CTC TTA TTT AAA GAT AGC CAG CTC R1 AAA TCT AGA ATC AAA TTC AAC GAA CAG ATA F2 AAA TCT AGA ATC AGC TTT CTA TCT ACA AGC R2 AAA GCA TGC ATG TTT GAT ATT GGC GTT AAT KF AAA TCT AGA ATG AGC CAT ATT CAA CGG GA KR AAA TCT AGA CCA ATT CTG ATT AGA AAA AC

<PCR reaction conditions> 94 ° C 5min 94 ° C 1 min 30 cycles 60 ° C 1 min 72 ℃ 1 min 72 ℃ 10 min

As a result of PCR using the above primer, rfaH Two 100 bp deficient PCR products were obtained (see Frag 1 and Frag 2, FIG. 1). Among them, Frag 1 was inserted into pGEM-T easy vector (Promega, Madison, USA) using SalI and XhoI, and sequencing analysis (Solgent Co. [Taejon, Korea]) was performed to confirm cloning. Frag2 was further inserted into the identified Frag1 + T-vector using XbaI and SphI, and a kanamycin cassette obtained by treating XbaI with pS43-Km3 was inserted between the fragments. The Frag1 + KM + Frag2 insert was then cloned into the pMEG375 vector, a donor vector, using SalI and SphI.

1-4. Screening process

The present inventors have found that the cloned Escherichia E. coli X7213 (Diaminopimelic (DAP) required) / pMEG375 (AMP) + Frag1 + KM + Frag2 and Salmonella Typhimurium conjugated and then the first selection in a DAP-free plate (with AMP) and the resulting colony was streaked on a new plate and separated. A completely isolated colony was cultured in LB liquid medium (without AMP) for 48 h and a second selection process was performed on LB + sucrose + KM plate. Thereafter, colonies generated on LB + sucrose + KM plates were again selected using M9 medium and LB medium. That is, a colony that grew in LB and did not grow in M9 was selected again.

Finally, 10 colonies selected were identified by PCR and found two ST- rfaH Del (M4, M8) deficient in rfaH gene. PCR was performed using KF and R2 primers to confirm the rfaH gene deletion of these strains. As a result, an expected PCR product of 1815 bp was obtained (FIG. 3).

The rfaH PCR was performed using F1 and KR primers to confirm the gene deletion, and the PCR product of the expected size of 2657 bp was confirmed. As a result of PCR using KF and R2 primers, the expected size of 1815 bp was confirmed (Fig. 4). Finally, through DNA sequencing, as shown in Fig. 1 as desired by the present inventors, rfaH It was confirmed that the gene was defective (Fig. 5).

< Example  2>

Wild type Mutant Growth rate  compare

The present inventors selected rfaH- deficient Salmonella typhimurium strains so as not to grow in the M9 medium but grow in the LB medium as described above. In other words, the mutant type of the gene related to the infectivity grows in the nutrient-rich medium, but in the minimum medium M9 medium, the aromatic amino acid or its precursor ( p- amino benzoic acid or 2, 3-dihydroxy benzoic acid), the mutant Salmonella typhimurium strain lacking the rfaH gene related to the infectivity was unable to grow in the M9 medium.

The present inventors tried to test the utility of the mutants as a vaccine through the growth phase of the strains.

Experimental methods were reference to Prior Art 1, that is, form the wild-type and rfaH mutant grown in LB medium Salmonella typhimurium gyunjueul respectively 1: and incubated stirring was inoculated in LB medium and M9 medium 1000 ratio (37 ℃, 230 rpm) . OD and CFU / ml were measured using a spectrometer (SmartSpec ™ 3000, Bio-Rad, USA) over time to determine the growth rate of the strain.

As a result, when comparing the growth rates in LB medium, rfaH The growth rate of the mutant strain was inhibited between 120 and 270 minutes compared with the wild type strain. These results were also more pronounced in strains grown in M9 medium (see FIG. 6).

< Example  3>

Wild type Mutant Infectivity  And Survival rate  compare

3-1. Infectivity  compare

The present inventors have found that, To investigate the possibility of using the rfaH mutant Salmonella typhimurium strain as a vaccine, we tried to compare cell infectivity.

That is, the present inventors constructed a mouse macrophage cell line, Raw 264.7 (ATCC) cells, in a 24-well plate at 5 × 10 ⁴ cells / well. DMEM containing 10% FBS (WISENT INC) Dulbeccos Modified Eagle Medium, Gibco).

The wild-type and mutant Salmonella typhimurium strains were all cultured on agar plates for 24 hours. The colonies were picked up with a sterilized toothpick, and then released into DMEM containing 10% FBS. The CFU was measured at OD 540 nm, 10 &lt; ⁷ &gt; CFU / ml.

The wells in which Raw 264.7 (ATCC) cells were cultured were washed twice with DMEM containing 10% FBS, 0.5 ml of the diluted Salmonella strain-containing medium was dispensed into each well, and 5% CO ₂ incubator without agitation. The medium was removed from all wells, washed twice with sterile PBS, and 0.5 ml of DMEM containing 100 μg / ml gentamicin and 10% FBS was added. After gentamicin treatment and 1 hour later, the cells were dissolved in 0.5 ml of distilled water and appropriately diluted and plated. On the other hand, the cells were continuously cultured in a medium containing gentamicin, and then they were dissolved in 0.5 ml of distilled water for 18 h or 24 h, and then appropriately diluted and plated.

As a result, it was found that rfaH It was confirmed that the cell survival rate was significantly decreased in the defective type (see FIG. 7A).

3-2. Cell survival rate comparison

The present inventors also sought to compare the cell viability of the wild-type and mutant Salmonella typhimurium strains in order to determine the possibility of using the vaccine of rfaH- deficient mutant Salmonella typhimurium strain.

To this end, the present inventors used the same method as in the prior art 4. First, mouse macrophage cell line Raw 264.7 (ATCC) was divided into 24-wells at ⁵ × 10 ⁵ cells / well and 10% FBS (WISENT INC) (Dulbeccos Modified Eagle Medium, Gibco). Both wild type and rfaH -deficient mutant strains were cultured at 37 ° C for 16 hours in stationary phase, washed with PBS, and diluted with DMEM medium. The diluted strains were added to the cells and allowed to enter the cells for 1 h. Then, the culture medium was removed from all wells, and the cells were washed twice with PBS. Then, 40 / ml gentamicin was added to kill remaining bacteria outside the cells. And 0.5 ml of DMEM containing 10% FBS. One hour after treatment with gentamicin, the medium was removed, washed with PBS, and treated with PBS containing 10% Triton X-100 for 2 minutes to release the bacteria present in the cells. The CFU of the released bacteria was appropriately diluted and plated to measure. Thereafter, in order to determine the intracellular replicon, cells were continuously cultured in a medium containing gentamicin, and treated with PBS containing 10% Triton X-100 for 2 minutes at 6 h or 24 h After dissolution, it was appropriately diluted and plated. The data were expressed in multiples based on the initial CFU values, and three independent experiments were performed.

As a result, we found that rfaH It was confirmed that the survival rate of the mutant type was significantly decreased (Fig. 7A), and the possibility of using the vaccine as the attenuated strain could be confirmed.

< Example  4>

Serum resistance, swarming mobility , Antimicrobial Peptides  Resistance comparison

4-1. Serum resistance

The present inventors To confirm the sensitivity of the immune system of the salmonella typhimurium strain deficient in rfaH in the human body, the serum resistance was examined.

For this experiment, we refer to the experimental method of the prior art 5. The cultivated wild type and rfaH deficient mutant strains on LB medium were each washed with saline and diluted with saline / cells / ml. The diluted strains (200 μl) were added to 48 wells and the same volume of 20% pooled human serum (Sigma) was added and incubated in a 37 ° C incubator for 1 hour. Thereafter, it was appropriately diluted and plated in a suitable medium, and the value was expressed in% as measured by CFU / ml.

As a result, it was confirmed that the wild type increased resistance to the immune system contained in human serum (100.3 ± 0.48), whereas the rfaH deficient mutant type had no colony formation and was extinguished by the immune system existing in human serum (See Table 3).

4-2. swarming mobility test

The present inventors have experimented with swarming mobility due to the fact that the synthesis of LPS present on the surface of bacteria is regulated by rfaH , which causes an immune response to humans and affects the swarming mobility of Salmonella.

For the experimental method, reference was made to Prior Art 5. Swarming motility was performed on LB solid plates containing 0.5% glucose (Sigma) and 0.6% agarose (Bacto ™). The wild-type and rfaH- deficient mutant Salmonella typhimurium strains previously grown on agar plates were washed and diluted with cells / ml with saline. 6 μl of the diluted strains were loaded into the center of the agar plate containing 0.5% glucose and 0.6% agarose in the form of droplets, and cultured in a 37 ° C incubator for 8 hours. The radius of the colonies was measured.

As a result, the present inventors showed that the wild type had a colony radius of 4.97 ± 0.15 cm, whereas the mutant type showed a 0.6 cm colony radius, indicating that the swaming mobility of the mutant type was significantly reduced compared to the wild type (Table 3).

4-3. Antimicrobial Peptides  Resistance comparison

The present inventors sought to compare the resistance of wild-type and mutant Salmonella typhimurium strains to polymyxin B, an antimicrobial peptide.

Polymyxin B is an antibiotic substance produced by aerobic bacteria in the soil. It has a polypeptide structure, has a narrow antibacterial spectrum and acts only on Gram-negative bacteria.

The present inventors performed this experiment by referring to the method described in the prior art document 5. Serial dilution of polymacin B (Sigma) from 0.1 to 100 mg / ml was followed by treatment with strains diluted to 10 ⁶ cells / ml, Lt; 0 &gt; C overnight. The optical density was measured using a conventional ELISA and the inhibition threshold value was set to 0.1 at OD ₅₅₀ . This evaluation was repeated three times.

As a result, the present inventors confirmed that the minimum inhibitory concentration (MIC) of the wild type was 4.8 ug / ml, which is significantly different from that of the mutant strain showing 0.64 ug / ml. In other words, it was confirmed that the mutant strains were much more vulnerable to the antimicrobial peptides than the wild type strains, which confirmed the possibility of using the mutant strain as a vaccine.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> KNU-Industry Cooperation Foundation          KONYANG UNIVERSITY INDUSTRIAL COOPERATION GROUP <120> rfaH mutated Salmonella typhimurium and a composition for food          poisoning prevention using the same <130> PN1209-459 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1242 <212> DNA <213> Artificial Sequence <220> <223> Mutated rfaH gene sequence which is aquired from samonella          typhimurium and mutated to contain restriction enzyme site and          kanamycin sequence in the middle of the gene <400> 1 atgcaatcct ggtatttact gtactgcaaa cgcgggcaac ttcagcgtgc tcaggaacac 60 ctcgaaagac aagcggtaag ttgcctgaca ccgatgatca ccctggaaaa aatggtacgc 120 ggaaaacgta ccttcgtcag cgaaccgctc tttcctaatt atctgttcgt tgaatttgat 180 tctagaatga gccatattca acgggaaacg tcttgctcga ggccgcgatt aaattccaac 240 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg 300 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa 360 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt 420 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc 480 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa 540 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat 600 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac 660 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc 720 tggaaagaaa tgcataagct tttgccattc tcaccggatt cagtcgtcac tcatggtgat 780 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga 840 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag 900 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg 960 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt ggtctagaca 1020 gctttctatc tacaagcccg aaggcgttgt cgatcctgaa accccctatc ccggcgatag 1080 cgtcatcatc acggaaggcg catttgaagg gctgaaagcg atttttaccg aaccggatgg 1140 cgaaacgcgt tcgatgttac tgcttaattt actcaataaa gaagtgaagc agagcgtaaa 1200 aaacaccggt tttcgcaaga tttaggtttt cgcaagattt ag 1242

Claims (10)

Salmonella typhimurium strain in which the gene rfaH has been mutated.
The method according to claim 1,
Mutated rfaH Wherein the nucleotide sequence of the gene is SEQ ID NO: 1. The method according to claim 1,
Wherein the Salmonella typhimurium strain is salmonella enterica serovar Typhimurium having a phage type of U302. The method according to claim 1,
The strain of Salmonella typhimurium is characterized in that the strain is attenuated. 5. The method of claim 4,
Wherein the attenuation is due to an increased susceptibility to antimicrobial peptides present in the cell. 6. The method of claim 5,
Wherein the antimicrobial peptide is Polymyxin B (Salmonella typhimurium). 7. A vaccine composition for preventing or treating food poisoning comprising the Salmonella typhimurium strain according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. (A) extracting genomic DNA from Salmonella typhimurium (phage type U302);
(B) amplifying the rfaH gene from the genomic DNA;
(C) cleaving a portion of the rfaH gene and inserting an antibiotic resistance marker;
(D) inserting the rfaH partial deletion gene into the vector;
(E) transforming said vector into E. coli;
(F) joining the transformed Escherichia coli with Salmonella typhimurium strain;
The Salmonella typhimurium mutant type of rfaH step (g) selecting a strain; rfaH method of producing a mutant type Salmonella typhimurium strain comprising a. 9. The method of claim 8,
Wherein a primer of SEQ ID NOS: 2 to 5 is used to delete a part of the rfaH gene, and a polymerase chain reaction is used to delete a part of the rfaH gene. 9. The method of claim 8,
Wherein said selecting step comprises selecting Salmonella typhimurium strains that survive in LB medium and kill in M9 medium. A method for producing a mutant Salmonella typhimurium strain.

Images