KR101965481B1 - Peptide for detecting shigella flexneri and salmonella enteritidis - Google Patents

Abstract

The present invention relates to a peptide that specifically binds to the outer membrane protein of Schizera flexineri or Salmonella enteritidis, a nucleic acid molecule encoding the same, a recombinant expression vector comprising a nucleic acid molecule, a transformant transformed with a recombinant expression vector, A kit comprising the peptide, and a method for detecting Shigella flexneri and Salmonella enteritidis using the peptide.
Peptides are easy to function due to their low molecular weight, and their manufacturing process is somewhat simple. In particular, the peptide of the present invention has a specific binding ability to the outer membrane protein of S. flexneri and S. enteritidis , and is effective for the detection of two pathogens and the diagnosis of diseases.

Description

Peptide for detecting shigella flexneri and salmonella enteritidis < RTI ID = 0.0 >

The present invention relates to a peptide that specifically binds to the outer membrane protein of Schizera flexineri or Salmonella enteritidis, a nucleic acid molecule encoding the same, a recombinant expression vector comprising a nucleic acid molecule, a transformant transformed with a recombinant expression vector, A kit comprising the peptide, and a method for detecting Shigella flexneri and Salmonella enteritidis using the peptide.

Livestock living in controlled livestock environments are always exposed to the risk of diseases related to environmentally harmful substances. In particular, when livestock is exposed to environmental pollution, toxic compounds and heavy metals for a long period of time, the substances absorbed into the body are not discharged to the outside of the body. In some cases, they remain in the body for several years and cause irreparable damage. It may accumulate in a large amount and adversely affect the livestock food safety. In order to select peptides as biomarkers capable of specifically detecting harmful microorganisms.

Salmonella spp. Is a pathogen of the common infectious disease that infects mammals, including humans, causing sepsis, diarrhea, and pneumonia. The transmission of Salmonella spp. Is infected with healthy animals and humans through food and polluted environment. The prevention of the treatment of livestock diseases and the public health of the people are becoming more important because of the increase in the number of resistant bacteria due to misuse and abuse of antibiotics. Salmonella is a pathogenic organism that can be present in humans, animals, and also in the environment. The major serotypes associated with food poisoning are S almonella typhimurium and Salmonella enteritidis .

Outer membrane proteins (OMPs) in cells are important molecules between cells and the environment. The outer membrane proteins are generally abundant and are used as good vaccine candidates because they are in direct contact with the host immune system. The outer membrane protein has been studied and successfully used as an antigenic potential of a vaccine against several bacterial infections.

Phage display is a technique that allows expression of an exogenous polypeptide on the surface of a phage particle, and a library of phage particles is used to select peptides that bind to a desired target material. Specific ligands or peptides isolated from phage display libraries can be used for drug design and vaccine development, and new peptides with novel amino acid sequences have been discovered and improved for use in early diagnosis of diseases.

The phage display technology, which is one of the widely used molecular evolutionary technologies, was used to target the outer membrane proteins of each of the pathogens S. flexneri and S. enteritidis to select peptides showing specific binding ability . This peptide has specific binding ability to the outer membrane proteins of S. flexneri and S. enteritidis . It is a biomarker for two pathogens. It can be used for the development of technology for the detection of target substances and diagnosis of disease, as well as for the development of therapeutic agents for various diseases. have.

It is an object of the present invention to provide a method for inhibiting the binding of one or more outer membrane proteins selected from the group consisting of Shigella flexneri and Salmonella enteritidis comprising the amino acid sequence of SEQ ID NO: Lt; / RTI > peptide.

Another object of the present invention is to provide a peptide that specifically binds to the outer membrane protein of Shigella flexneri , which is composed of the amino acid sequence of SEQ ID NO: 2.

Another object of the present invention is to provide a peptide which specifically binds to the outer membrane protein of Salmonella enteritidis , which is composed of the amino acid sequence of SEQ ID NO: 3.

It is still another object of the present invention to provide a nucleic acid molecule encoding the amino acid sequence of the peptide of any one of the above-mentioned claims.

It is still another object of the present invention to provide a recombinant expression vector comprising the nucleic acid molecule.

It is intended to provide a transformant transformed with another expression vector of the present invention.

Another object of the present invention is to provide a composition for detecting Salmonella enteritidis comprising Shigella flexneri or Salmonella enteritidis including the aforementioned peptide.

Another object of the present invention is to provide a kit for detecting Shigella flexneri or Salmonella enteritidis comprising a peptide.

Yet another object of the present invention is to provide a method for detecting a protein to be detected, comprising: (a) contacting a sample to be detected with a peptide of any one of SEQ ID NOS: 1 to 3; And (b) Shigella flexneri comprising the step of confirming the binding of the peptide to Salmonella enteritidis, which may be contained in a sample to be detected, with Shigella flexneri or Salmonella enteritidis , or And a method for detecting Salmonella enteritidis .

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition comprising at least one kind of outer membrane protein selected from the group consisting of Shigella flexneri and Salmonella enteritidis, which is composed of the amino acid sequence of SEQ ID NO: Can be provided.

The present invention can also provide a peptide that specifically binds to the outer membrane protein of Shigella flexneri , which is composed of the amino acid sequence of SEQ ID NO: 2.

The present invention can also provide a peptide that specifically binds to the outer membrane protein of Salmonella enteritidis , which is composed of the amino acid sequence of SEQ ID NO: 3.

The amino acid sequences of SEQ ID NOS: 1 to 3 of the present invention were found to identify peptide sequences that specifically bind to the outer membrane protein of Shigella flexneri or Salmonella enteritidis using a phage display . The peptide according to the present invention is advantageous in that it is relatively easy to mass-produce and less toxic than an antibody. It is also known that Shigella flexneri and Salmonella enteritidis It has a high binding strength and does not undergo denaturation during thermal / chemical treatment. Moreover, because of its small molecular size, it can be used as a fusion protein by binding to other proteins. The peptides of the present invention can be produced by chemical synthesis known in the art. Representative methods include, but are not necessarily limited to, liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemistry. The peptides of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is prepared according to a conventional method. DNA sequences can be prepared by PCR amplification using appropriate primers. Alternatively, DNA sequences can be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (e.g., marketed by Biosearch or Applied Biosystems). The constructed DNA sequence is operatively linked to this DNA sequence and contains one or more expression control sequences (e.g., promoters, etc.) that regulate the expression of the DNA sequence , And the host cells are transformed with the recombinant expression vector formed therefrom. The resulting transformant is cultured under appropriate medium and conditions so that the DNA sequence is expressed, and the substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery can be performed using methods known in the art (e.g., chromatography). By "substantially pure peptide" in the above, it is meant that the peptide according to the present invention does not substantially contain any other protein derived from the host. The peptides of the present invention are capable of known denaturation for eliminating the reactivity of the amino terminal at the N-terminus, for example, but are not limited to, acetylation.

The target of the peptide of the present invention, " Shigella flexneri " and Salmonella enteritidis , correspond to strains of representative Shigella species and Salmonella species.

In particular, peptides recognizing the outer membrane proteins of Shigella flexneri and Salmonella enteritidis, which are the livestock disease inducing microorganisms, can be used as biomarker proteins for detecting harmful microorganisms as new materials and can be used not only in medical fields but also in agricultural, .

According to one embodiment of the present invention, there is provided a nucleic acid molecule encoding an amino acid sequence of a peptide. The term " nucleic acid molecule " in the present invention encompasses both DNA (gDNA and cDNA) and RNA molecules. The nucleotide as a basic constituent unit in the nucleic acid molecule includes not only natural nucleotides but also analogs and an analogue. The sequence of the nucleic acid molecule encoding the peptide of the present invention can be modified. Such modifications include addition, deletion, or non-conservative substitution or conservative substitution of nucleotides.

The nucleotides of the present invention are also interpreted to include nucleotide sequences that exhibit substantial identity to the nucleotide sequences described above. The above-mentioned substantial identity is determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. % Homology, at least 90% homology in one particular example, and at least 95% homology in another specific example.

Another embodiment of the present invention provides a recombinant expression vector comprising the nucleic acid molecule. The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this can be found in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts. It is preferable that the nucleic acid molecule of the present invention is derived from a prokaryotic cell and that the prokaryotic cell is used as a host in consideration of the convenience of cultivation.

For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (such as a tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL 貫 promoter, pR 貫 promoter , rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation and a transcription / translation termination sequence. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158: 1018-1024 (1984)) and the left promoter of phage l The promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980) can be used as the regulatory region.

The vectors that can be used in the present invention include plasmids such as pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19 and pET which are frequently used in the art, phages such as λgt4 · λB, ,?? z1, and M13) or a virus (e.g., SV40 or the like).

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from a genome of a mammalian cell (for example, a metallothionein promoter) or a mammalian virus Virus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be fused with other sequences to facilitate the purification of oligopeptides that specifically bind to Salmonella enteritidis and Shigella flexneri expressed therefrom. Fusion sequences include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA) Is 6x His. Because of the additional sequence for such purification, proteins expressed in the host are rapidly and easily purified through affinity chromatography.

According to a preferred embodiment of the present invention, the fusion protein expressed by the vector containing the fusion sequence is purified by affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate of the enzyme, can be used. When 6xHis is used, Ni-NTA His-bonded resin column (Novagen, USA) Oxirredoxin can be obtained quickly and easily.

On the other hand, the expression vector of the present invention includes, as a selection marker, an antibiotic resistance gene commonly used in the art and includes, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, There is a resistance gene for mitine and tetracycline.

The present invention can also provide a transformant transformed with an expression vector. The host cell which can stably and continuously clone and express the vector of the present invention can be any host cell known in the art such as E. coli Origami2, E. coli JM109, E. coli BL21 (DE3 ), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli strains such as E. coli strain, Bacillus subtilis, Bacillus strains such as Bacillus strain, and Salmonella typhimurium, Serratia marcesensis, and various enterococci such as Pseudomonas species and strains.

In addition, Saccharomyce cerevisiae, insect cells and human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and the like can be used.

Methods of delivering a vector of the present invention into a host cell include the CaCl2 method (Cohen, SN et al. Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)) when the host cell is a prokaryotic cell, 16: 557-580 (1983), and Hanahan, D., J. MoI. Biol., 16: 557-580 ) And electroporation (Dower, WJ et al., Nucleic Acids Res., 16: 6127-6145 (1988)). When the host cell is a eukaryotic cell, the microinjection method (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al. Virology, 52: 456 Liposome-mediated transfection (Wong, TK et al., Gene, 10: 87 (1980)), DEAE-dex (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)), and Tran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 The vector can be injected into the host cell.

The vector injected into the host cell can be expressed in host cells and in this case a peptide specifically binding to a large amount of Shigella flexneri or Salmonella enteritidis is obtained. For example, when the expression vector comprises a lac promoter, the host cell may be treated with IPTG to induce gene expression.

The present invention can also provide a composition for detecting Shigella flexneri or Salmonella enteritidis comprising a peptide of any one of SEQ ID NO: 1 to SEQ ID NO: 3.

In order to easily identify, detect and quantify whether the peptide of the present invention has formed a bond with Shigella flexneri or Salmonella enteritidis, the peptide of the present invention is provided in a labeled state . I.e., linked (e.g., covalently bonded or bridged) to a detectable label. The link may be formed directly between the peptide of the present invention and a detectable label, but may also be formed via a link. The linker can be any known in the art, and the sequence of a suitable peptide linker can be selected in consideration of the following factors: (a) the ability to be applied to a flexible extended conformation; (b) the ability to not create a secondary structure that interacts with the epitope; And (c) the absence of a hydrophobic moiety or moiety having charge capable of reacting with the epitope.

The peptide of the present invention may be labeled with one selected from the group consisting of chromogenic enzymes, radioactive isotopes, chromophores, luminescent materials and fluorescent materials.

Specifically, the label may be, for example, peroxidase, alkaline phosphatase corresponding to a chromogenic enzyme; 124I, 125I, 111In, 99mTc, 32P, 35S corresponding to the radioisotope; Chromophore; And may be, for example, FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, and quantum dots, which correspond to a luminescent material or a fluorescent material. It does not.

Similarly, the detectable label may be an antibody epitope, a substrate, a cofactor, an inhibitor or an affinity ligand. Such labeling may be performed during the synthesis of the peptide of the present invention, or may be performed in addition to the peptide already synthesized.

The oligopeptide comprising the detectable label can be detected using conventionally known detection methods for each label. Specifically, for example, when the fluorescence signal FITC is used as a label, the fluorescence intensity for each peptide concentration at a maximum wavelength (excitation: 495 nm, emission: 520 nm) of the FITC connected to the peptide using a fluorescence spectrophotometer A measurement method can be used.

A kit for detecting Shigella flexneri or Salmonella enteritidis comprising the peptide of any one of SEQ ID NOS: 1 to 3 can be provided. The Shigella flexneri or Salmonella enteritidis detection kit of the present invention may comprise a peptide of SEQ ID NO: 1 to SEQ ID NO: 3 or a nucleic acid molecule encoding the same, wherein the peptide or nucleic acid The molecule may be one obtained by chemical or genetic engineering methods as described above. The above peptides are the same as described above for explaining other aspects of the present invention. The kit of the present invention may further comprise a buffer solution or a reaction solution that stably maintains the structure or physiological activity of the peptide. Further, in order to maintain stability, it may be provided in a powder state or in a state dissolved in an appropriate buffer or maintained at 4 캜.

The Shigella flexneri or Salmonella enteritidis detection kit according to the present invention can be quickly and conveniently diagnosed on the spot as compared with the conventional PCR method and can be used for detecting Shigella flexneri or Salmonella enteritidis ( Salmonella enteritidis) . ≪ / RTI > It is possible to increase sensitivity to food poisoning bacteria by connecting the peptides of the present invention to other known probes and to detect only food poisoning bacteria or alien bacteria specifically and to detect food poisoning bacteria or dysentery bacteria by fluorescence microscopy Lt; / RTI > system.

In order to facilitate detection of Shigella flexneri or Salmonella enteritidis, the peptides included in the detection kit of the present invention may be provided in a labeled state as described above. On the other hand, when the peptide of the present invention is provided without being labeled, the Shigella flexneri or Salmonella enteritidis detection kit according to the present invention is contained in the in vitro or detection target of the peptide of the present invention And may further include components for searching for the presence or absence of binding to Salmonella enteritidis strain Shigella flexneri or Salmonella enteritidis . The components may be known compounds for the labeling of the peptides of the invention or may be conjugated to a peptide of the invention or an antibody against a particular receptor that binds to the peptide of the invention or a secondary antibody thereto, And may be a reagent for detection thereof. Specifically, the diagnosis of the present invention can be carried out by modifying a conventional immunoassay method or protocol. For example, the diagnosis of the present invention can be carried out by using the peptide of the present invention instead of the antibody in the conventional immunoassay, and the process is the same. For example, an enzyme-linked immunosorbant assay (ELISA) is performed with a slight modification to an enzyme-linked oligonucleotide assay (ELONA).

In addition, the peptide of the diagnostic kit of the present invention may be provided in a form coated on the surface of the plate. In this case, the target sample is inoculated directly to the plate, reacted under appropriate conditions, and then the plate of Shigella flexneri or Salmonella enteritidis contained in the target sample on the surface of the plate, By observing the binding, it is possible to detect Shigella flexneri or Salmonella enteritidis .

The Shigella flexneri or Salmonella enteritidis detection kit of the present invention can be used in a form such as a bottle, a tub, a sachet, an envelope, a tube, an ampoule, Which may be partially or wholly formed from plastic, glass, paper, foil, wax, and the like. The container may be part of it or may be fitted with a fully or partially detachable cap which may be attached to the container by mechanical, adhesive or other means. The container may also be equipped with a stopper accessible to the contents by the injection needle.

A method for detecting Salmonella enteritidis or Shigella flexneri including the following steps can be provided.

(a) contacting a sample to be detected with a peptide of any one of SEQ ID NOS: 1 to 3; And

(b) confirming whether the peptide binds to Shigella flexneri or Salmonella enteritidis which can be contained in the sample to be detected.

The " sample to be detected " of the present invention means a sample which is likely to be present in the presence of Shigella flexneri or Salmonella enteritidis and whose presence needs to be confirmed. Specifically, it may be collected from, for example, at least one of liquid, soil, air, food, feed, waste and plants and animals, but is not limited thereto. More specifically, the liquid may be water, blood, urine, tears, sweat, saliva, lymph and cerebrospinal fluid, and the water includes precipitation, seawater, lake water and rainwater. And the waste includes sewage, wastewater, etc., and the animal or plant includes a human body. In addition, the animal or plant animal is not particularly limited as long as it provides a tissue that can be used for food, feed for plants and animals, and the like.

The confirmation of the binding in step (b) of the present invention can be referred to the above for the composition for detecting Shigella flexneri or Salmonella enteritidis, which is another embodiment of the present invention, Omission is avoided in order to avoid the excessive complexity described herein.

Peptides are easy to function due to their low molecular weight, and their manufacturing process is somewhat simple. In particular, the peptide of the present invention has a specific binding ability to the outer membrane protein of S. flexneri and S. enteritidis , and is effective for the detection of two pathogens and the diagnosis of diseases.

FIG. 1 is a diagram illustrating a biopanning process for peptide detection using a phage library.
FIG. 2 shows the result of confirming the template DNA extracted in the third round through PCR using agarose electrophoresis; (A) Template DNA extracted from library phage specifically binding to the outer membrane protein of S. flexneri, (B) Template DNA extracted from library phage specifically binding to the outer membrane protein of S. enteritidis.
FIG. 3 shows the result of confirming the template DNA extracted in 4 rounds by PCR through agarose electrophoresis; (A) Template DNA extracted from library phage specifically binding to the outer membrane protein of S. flexneri, (B) Template DNA extracted from library phage specifically binding to the outer membrane protein of S. enteritidis.
FIG. 4 shows the result of confirming template DNA extracted in 5 rounds through PCR using agarose electrophoresis; (A) Template DNA extracted from library phage specifically binding to the outer membrane protein of S. flexneri, (B) Template DNA extracted from library phage specifically binding to the outer membrane protein of S. enteritidis.
FIG. 5 is a phage single DNA sequence analysis for Shigella flexneri and Salmonella enteritidis ; (A) Sequence analysis for Shigella flexneri , (B) Sequence analysis for Salmonella enteritidis .

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1. Shigella flexneri Wow Salmonella enteritidis Isolation of the outer membrane protein

In this example, the outer membrane proteins of S. flexneri and S. enteritidis, which are harmful microorganisms of livestock diseases, were separated and the separation method was as follows. ReadyPrep ^TM Protein Extraction Kit (Bio-Rad Laboratories, Inc.) was used to isolate the outer membrane protein. Two kinds of pathogens were cultured at 37 ° C for 12 hours and then centrifuged at 3000 rpm for 15 minutes. Then M1 buffer (lysis buffer) and 0.1 mM PIC (protease inhibitor cocktails) provided in the kit were mixed with the precipitate, Crush the precipitate using a sonicator. The milled precipitate was mixed with M2 buffer (1: 1), vortexed for 1 minute and ice for 1 minute. After 4 ~ 5 times, it was reacted in ice for 10 minutes and reacted at 37 ℃ for 30 minutes . Separate centrifuge at 13000 rpm for 5 minutes to remove supernatant and collect precipitate. Repeat this process two more times to include more pure outer membrane proteins in the precipitate.

Example 2. Phage display S. flexneri Wow S. enteritidis Of peptides specifically binding to the outer membrane protein of

In this example, peptides that specifically bind to the outer membrane proteins of S. flexneri and S. enteritidis were screened using a phage display library, and the selection procedure was as follows (see FIG. 1). The outer membrane protein isolated in Example 1 was fixed on a plate, mixed with phage library, adjusted to 1 ml with TBST buffer, and reacted at room temperature for 60 minutes. One milliliter of elution buffer (0.2 M Glycine-HCl, pH 2.2) was added to each plate for 15 min at room temperature to screen peptides that specifically bind to each outer membrane protein. The reaction solution containing the phage library containing the separated specific peptides was recovered and neutralized by adding 150 μl of 1 M Tris-HCl (pH 9.1) buffer. 800 μl of the separated library phage was inoculated into E. coli ER2738, mixed with 20 ml of LB culture, and incubated at 37 ° C for 4.5 hours for amplification. The reaction solution was centrifuged at 12,000 g at 4 ° C to recover 80% of the supernatant, and 20% PEG / 2.5M NaCl was mixed and reacted at 4 ° C for 12 hours to precipitate a library phage containing a specific peptide , And centrifuged at 12,000 g at 4 ° C to recover only the library phage containing the specific peptide. Afterwards, the phage display library proceeded up to five times. As the round of phage display library proceeded, the reaction conditions were tightened to obtain a peptide that specifically binds to the outer membrane proteins of S. flexneri and S. enteritidis as target substances .

Example 3. Extraction of specific peptides in selected library phage

10 to 15 library phages corresponding to 3, 4, and 5 rounds were collected for specific peptide screening, and the method for extracting peptides in the library phage containing the recovered specific peptides was as follows. E. coli ER2738 was inoculated into 1 ml of LB culture at a ratio of 1: 100, followed by incubation at 37 ° C for 5 hours. After the incubation, the mixture was centrifuged at 14,000 rpm for 1 minute, and 500 μl of the supernatant and 200 μl of 20% PEG / 2.5M NaCl buffer were mixed and reacted at room temperature for 15 minutes. The reaction solution was centrifuged at 14000 rpm for 10 minutes at 4 ° C to remove the supernatant, and only the peptide was isolated. The peptide obtained by centrifugation was washed with ethanol to remove impurities and obtain pure peptides. 100 ㎕ of Iodide buffer was added to the obtained peptide, mixed with 200 ㎕ ethanol, and reacted at room temperature for 15 minutes. The reaction solution was centrifuged at 14,000 rpm for 10 minutes at 4 ° C to remove the supernatant, and the peptide was washed with 500 μl of 70% ethanol. The supernatant was completely removed by centrifugation once more, After drying for a few minutes, ethanol was completely volatilized. Then, 30 μl of TE buffer was added to the washed peptide.

Example 4. S. flexneri Wow S. enteritidis Identification of peptides that specifically bind to the outer membrane proteins of

A pair of primers was synthesized from bioneer (Bioneer, Korea) to amplify peptides (DNA oligonucleotides) that specifically bind to the outer membrane proteins of S. flexneri and S. enteritidis [forward primer: 5'-CCGATTCCTTTAGTGGTACCTTTCTAT -3 '(SEQ ID NO: 4), reverse primer 5'-CCCTCATAGTTAGCGTAACG-3' (SEQ ID NO: 5)]. The DNA oligonucleotide was amplified by PCR using the above primers. The reaction composition was 5 μl of template DNA, 5 μl of 10 × PCR buffer solution, 4 μl of dNTP mixture, 2 μl of 10 μM forward primer, 2 μl of 10 μM reverse primer , 0.2 μl (1 unit / μl) of Ex Taq polymerase (Takara, Japan) and 31.8 μl of distilled water. The reaction conditions for the amplification of landen template DNA were denaturation at 95 ° C for 4 minutes, 30 cycles of reaction at 95 ° C for 30 seconds, 42.5 ° C for 30 seconds, and 72 ° C for 30 seconds, followed by addition at 72 ° C for 7 minutes Was used. After the PCR reaction, 4 를 was taken and the amplified product was confirmed by 0.7% agarose gel electrophoresis. The amplified product was purified using a PCR purification kit (GeneAll, Korea) and recovered in 30 쨉 l of distilled water 3, Fig. 4).

Primer sequence Phage-F CCG ATT CCT TTA GTG GTA CCT TTC TAT Phage-R CCC TCAT AGT TAG CGT AACG

All of the above genes were purified using GeneAll PCR DNA purification kit.

Example 5. Specially Binding Peptide Sequence Analysis

Using the outer membrane protein acting as an antigen recognition site, a peptide sequence specifically binding to the outer membrane protein of S. flexneri and S. enteritidis isolated in Example 1 was screened, and the specificity obtained in Examples 2 and 3 Peptides displaying binding affinity were selected and extracted, and sequence analysis (Cosmogenetech, Korea) of peptides (DNA oligonucleotides) amplified and recovered in Example 4 was carried out.

(SEQ ID NO: 1), characterized in that it binds commonly to the outer membrane proteins of S. flexneri and S. enteritidis through sequencing, a peptide which specifically binds to the outer membrane protein of S. flexneri No. 2) and the outer membrane protein of S. entertidis (SEQ ID NO: 3) (see FIG. 5).

<110> CHONBUK UNIVERSITY INDUSTRIAL COOPERATION CORP <120> Peptide for detecting shigella flexneri and salmonella          enteritidis <130> 1061588 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Shigella and Salmonella specific peptide <400> 1 Met His Pro Asn Ala Gly His Gly Ser Leu Met Arg   1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Shigella-specific peptide <400> 2 Tyr Leu Asp Tyr Val Ala Thr Ser Ser His Lys Tyr   1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Salmonella-specific peptide <400> 3 Ser Ser Thr Leu Leu Asn Val Val Pro Lys Leu His   1 5 10 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplification of Salmonella and Shigella <400> 4 ccgattcctt tagtggtacc tttctat 27 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplification of Salmonella and Shigella <400> 5 ccctcatagt tagcgtaacg 20

Claims (10)

delete delete A peptide that specifically binds to the outer membrane protein of Salmonella enteritidis , which is composed of the amino acid sequence of SEQ ID NO: 3. A nucleic acid molecule encoding the amino acid sequence of the peptide of claim 3. A recombinant expression vector comprising the nucleic acid molecule of claim 4. A transformant transformed with the recombinant expression vector of claim 5. A composition for detecting Salmonella enteritidis comprising the peptide of claim 3. [Claim 7] The composition according to claim 7, wherein the peptide is labeled with at least one selected from the group consisting of a chromogenic enzyme, a radioisotope, a chromopore, a luminescent material and a fluorescent material. A kit for detecting Salmonella enteritidis comprising the peptide of claim 3. A method for detecting Salmonella enteritidis comprising the steps of:
(a) contacting a sample to be detected with a peptide according to claim 3; And
(b) confirming whether the peptide is bound to Salmonella enteritidis that can be contained in the sample to be detected.

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