KR101729469B1 - Peptide Probe for Detection of Salmonella Enteritidis - Google Patents

Abstract

The present invention relates to a peptide probe that specifically binds to Salmonella enteritidis KCCM 12021. Using the peptide probe of the present invention, Salmonella enteritidis KCCM 12021 can be effectively and specifically detected.

Description

 [0001] The present invention relates to a peptide probe for detection of Salmonella enteritidis (Salmonella Enteritidis)

The present invention relates to a peptide probe that specifically binds to Salmonella enteritidis KCCM 12021.

Salmonella is a type of proteobacteria belonging to the Salmonella family of enterobacteria. It is a rod-shaped bacterium with a diameter of about 0.7-1.5 μm and a length of about 2-5 μm. It lives mainly in human or animal digestive tracts.

Salmonella infection usually causes headache and fever, severe abdominal pain, diarrhea, nausea and vomiting. Salmonella is transmitted to humans mainly when consuming less cooked food using infected livestock (ie meat, poultry, eggs and their by-products) as food. Salmonella is also transmitted from person to person through the hands of an infected person, and from animals to humans.

Salmonella is the most common disease caused by food poisoning. Foodborne pathogens are a number of pathogens known to be caused by pathogenic bacteria, toxins expressed in pathogenic bacteria, viruses and toxins present in nature, but there are many causes of unknowns. According to the statistics of onset caused by food poisoning bacteria in Korea, the number of cases and the number of cases caused by pathogenic Escherichia coli and Salmonella have occupied the first and second places (refer to FIG. 8).

Currently, most methods for detecting food poisoning bacteria such as Salmonella and pathogenic Escherichia coli use cell culture method, PCR method, and immunoassay method (ELISA), and these methods suggest self-high sensitivity and specificity, Time is longer than that proposed, separation of the target molecule is necessary, and sometimes expensive detection equipment is necessary. Therefore, it is considered that there is a limit to detect foodborne pathogens early in the convenient way in the field.

It is very difficult to diagnose the disease within a short period of time when symptoms of disease do not appear due to the nature of food poisoning bacteria. It is essential to develop a detection method with high sensitivity and promptness for ultra - precision quarantine. Therefore, it is necessary to develop technology that can diagnose and detect early detection of Salmonella and pathogenic Escherichia coli, which occupy the first and second place, of foodborne pathogens.

Korean Patent Laid-Open Publication No. 10-2013-0062276 discloses a nucleic acid probe for detecting and / or quantifying Salmonella subsp. Korean Patent Laid-Open Publication No. 10-2013-0055040 discloses a nucleic acid probe for detecting Salmonella enteritidis , , Salmonella typhimurium, Salmonella gallinarum , and Salmonella pullorum . The present invention relates to a primer set for simultaneous detection of Salmonella typhimurium, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum . In addition, Japanese Laid-Open Patent Publication No. 10-2014-0075896 discloses a DNA aptamer specifically binding to Salmonella enteritidis.

The present invention relates to a peptide probe having a novel amino acid sequence not previously known, which specifically binds to Salmonella enteritidis KCCM 12021.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop a novel probe specifically binding to Salmonella enteritidis KCCM 12021 strain. As a result, a peptide specifically binding to the Salmonella enteritidis KCCM 12021 strain was identified, and it was found that Salmonella enteritidis KCCM 12021 strain can be specifically detected using the peptide.

Accordingly, an object of the present invention is to provide an oligopeptide which specifically binds to Salmonella enteritidis KCCM 12021.

It is still another object of the present invention to provide a nucleic acid molecule which encodes the amino acid sequence of the above-mentioned oligopeptide.

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

It is still another object of the present invention to provide a transformant which is transformed with the above-mentioned expression vector.

It is still another object of the present invention to provide a composition for detecting Salmonella enteritidis KCCM 12021 comprising the above-described oligopeptide.

It is still another object of the present invention to provide a kit for detecting Salmonella enteritidis KCCM 12021 comprising the above oligopeptide.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided an oligopeptide specifically binding to Salmonella enteritidis KCCM 12021 having the amino acid sequence of the first sequence of the sequence listing.

The present inventors have made extensive efforts to develop a novel probe specifically binding to Salmonella enteritidis KCCM 12021 strain. As a result, a peptide specifically binding to Salmonella enteritidis KCCM12021 was identified, and it was found that Salmonella enteritidis KCCM 12021 strain can be specifically detected.

The amino acid sequence of the first sequence of the sequence listing of the present invention is a random phage peptide library (phage peptide library constructed to have 2.7 billion different amino acid sequences through a random arrangement consisting of 12 amino acids fused to the M13 phage gp3 minor coat protein, Ph.D ^TM using (Phage display peptide Library Kit, New England Biolabs using purchase a) will find the oligopeptide sequence which specifically binds to Salmonella Entebbe utility discharge KCCM 12021 strain. the term on the terms "oligopeptide" Refers to a relatively small peptide generally consisting of about 10 amino acids before and after the amino acid, and more specifically, refers to a peptide composed of 12 amino acids constituting the above-mentioned peptide library. The "oligopeptide"Quot; peptide probe " The oligopeptide according to the present invention is advantageous in that it is relatively easy to mass-produce and has little toxicity as compared with the antibody. The oligopeptide of the present invention has an advantage of high binding strength to Salmonella enteritidis KCCM 12021 strain, The oligopeptide of the present invention can be prepared by chemical synthesis well known in the art. The oligopeptide of the present invention can be produced by chemical synthesis known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemistry. The peptides of the present invention can also be prepared by genetic engineering methods. A DNA sequence encoding the peptide is prepared according to a conventional method. The DNA sequence is amplified using appropriate primers There the DNA sequence using the (to sell from Biosearch or Applied Biosystems, Inc. example) may be synthesized W can be produced by PCR amplification by standard methods known in the art, for example, automatic DNA synthesizer in a different way. The constructed DNA sequence is operatively linked to the DNA sequence and contains one or more expression control sequences (e.g., promoters, enhancers, 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" herein is meant that the peptide according to the invention is substantially free of any other proteins derived from the host. Genetic engineering methods for peptide synthesis of the present invention can be found in the following references: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif., 1991; And Hitzeman et al., J. Biol. Chem., 255: 12073-12080, 1990. The peptides of the present invention are capable of known denaturation for eliminating the reactivity of the amino terminal of the N-terminus, for example, acetylation, no.

The target of the oligopeptide of the present invention, "Salmonella enteritidis KCCM 12021 ", corresponds to a strain of representative Salmonella species.

According to another aspect of the present invention, there is provided a nucleic acid molecule characterized by encoding an amino acid sequence of an oligopeptide.

As used herein, the term "nucleic acid molecule" is intended to encompass DNA (gDNA and cDNA) and RNA molecules in a comprehensive sense. Nucleotides which are basic constituent units in nucleic acid molecules include not only natural nucleotides but also analogs (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

Variations in nucleotides do not cause changes in the protein. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (e.g., by codon degeneration, six codons for arginine or serine), or codons that encode biologically equivalent amino acids ≪ / RTI >

Also, variations in the nucleotides may result in changes in the oligopeptides of the Salmonella enteritidis KCCM 12021 tablets of the present invention. Even when the oligopeptide of the present invention is a mutation that causes a change in the amino acid, it can be obtained that exhibits almost the same activity as the oligopeptide specifically binding to Salmonella enteritidis KCCM 12021 of the present invention.

It will be apparent to those skilled in the art that biological function equivalents that may be included in the oligopeptide specific binding to Salmonella enteritidis KCCM 12021 of the present invention will be limited to variations in the amino acid sequence exhibiting equivalent biological activity with the peptides of the present invention Do.

Such amino acid variations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are both positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In introducing the mutation, the hydrophobic index of the amino acid can be considered. Each amino acid is assigned a hydrophobic index according to its hydrophobicity and charge: isoruicin (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in imparting the interactive biological function of peptides. It is known that substitution with an amino acid having a similar hydrophobicity index can retain similar biological activities. When a mutation is introduced with reference to a hydrophobic index, substitution is made between amino acids showing a hydrophobic index difference preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that the substitution between amino acids with similar hydrophilicity values leads to peptides with homogeneous biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Aspartate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoru Isin (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When a mutation is introduced with reference to the hydrophilicity value, the amino acid is substituted preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

Amino acid exchange in peptides that do not globally alter the activity of the molecule is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

The oligopeptide specifically binding to Salmonella enteritidis KCCM 12021 of the present invention or the nucleic acid molecule for peptide coding may exhibit substantial identity with the sequence set forth in the sequence listing, Sequence is also included. The above-mentioned substantial identity is determined by aligning the above-described 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, at least 80% Homology, more preferably 90% homology, and most preferably 98% homology. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv. Appl. Math. 2: 482 (1981) Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from National Center for Biological Information (NBCI) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to another aspect of the present invention, there is provided a recombinant expression vector comprising the above-described 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 are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001) This document 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 the prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (such as a tac promoter, lac promoter, lac UV5 promoter, lpp promoter, pL ^lambda promoter, it is common to include the ^λ pR promoter, rac 5 promoter, amp promoter, recA promoter, SP6 Pro meoteo, trp promoter and T7 promoter, etc.), lie bojom binding site and a transcription / translation termination sequences for the initiation of the decryption. 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 phage left promoter ^lambda promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet. , 14: 399-445 (1980)).

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 an oligopeptide that specifically binds to Salmonella enteritidis KCCM 12021 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.

According to another aspect of the present invention, there is provided a transformant characterized by being transformed with the above-mentioned 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 strain, Bacillus subtilis, Bacillus strains such as Bacillus Chuo ringen systems such as E. coli W3110, 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.

The method of delivering the vector of the present invention into a host cell may be carried out by the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1973) , Hanahan, D., J. MoI. Biol. , 166: 557-580 (1997)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1983)) and electroporation (Dower, WJ et al., Nucleic Acids Res. , 16: 6127-6145 (1988)). In addition, when the host cell is a eukaryotic cell, microinjection (Capecchi, MR, Cell , 22: 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology , 52: 456 electroporation (Neumann, E. et al, EMBO J., 1:. 841 (1982)), liposome-mediated transfection method (Wong, TK et al, Gene , 10:87 (1980).), DEAE- (Yang et al., Proc. Natl. Acad. Sci. , 87: 9568-9572 (1990)) and dextran treatment (Gopal, Mol. Cell Biol. , 5: 1188-1190 ) Or the like into the host cell.

The vector injected into the host cell can be expressed in the host cell and in this case an oligopeptide specifically binding to a large amount of Salmonella enteritidis KCCM 12021 is obtained. For example, when the expression vector comprises a lac promoter, the host cell may be treated with IPTG to induce gene expression.

According to another aspect of the present invention, there is provided a composition for detecting Salmonella enteritidis KCCM 12021 comprising the above-described oligopeptide.

In order to easily identify, detect and quantify whether or not the oligopeptide of the present invention binds to Salmonella enteritidis KCCM 12021, the peptide of the present invention can be 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.

In one embodiment of the present invention, the oligopeptide of the present invention is 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.

According to another aspect of the present invention, there is provided a kit for detecting Salmonella enteritidis KCCM 12021 comprising the above-described oligopeptide. The Salmonella enteritidis KCCM 12021 detection kit of the present invention may comprise an oligopeptide of the first sequence or a nucleic acid molecule encoding the oligopeptide, wherein the oligopeptide or nucleic acid molecule is chemically or genetically engineered May be obtained. The oligopeptide is the same as described above for the explanation of 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 Salmonella typhimurium KCCM 11806 detection kit of the present invention can be provided in the form of a food poisoning bacteria fluorescence detection kit including Salmonella typhimurium KCCM 11806 which can be quickly and conveniently diagnosed in situ in comparison with the conventional PCR method. A system capable of increasing sensitivity to food poisoning bacteria by linking the oligopeptides of the present invention to other probes known in the art and capable of specifically detecting food poisoning bacteria and detecting food poisoning bacteria through fluorescence microscopy And the like.

In order to facilitate detection of Salmonella enteritidis KCCM 12021, oligopeptides included in the detection kit of the present invention may be provided in the labeled state as described above. Meanwhile, when the oligopeptide of the present invention is provided without labeling, the Salmonella enteritidis KCCM 12021 detection kit of the present invention can be used to detect the binding of the oligopeptide of the present invention to the Salmonella enteritidis strain And / or < / RTI > 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).

The oligopeptide 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 the binding of the oligopeptide to Salmonella enteritidis KCCM 12021 contained in the target sample on the surface of the plate is observed to detect the Salmonella enteritidis strain can do.

The Salmonella enteritidis KCCM 12021 detection kit of the present invention may take the form of a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., , 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.

According to another aspect of the present invention, there is provided a method for detecting Salmonella enteritidis KCCM 12021 comprising the steps of:

(a) contacting the oligopeptide of claim 1 with a sample to be detected; And

(b) confirming whether or not the oligopeptide binds to Salmonella enteritidis KCCM 12021 that can be contained in the detection target sample.

The "sample to be detected" of the present invention means a sample to which the presence of Salmonella enteritidis KCCM 12021 is likely to be present. 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.

Confirmation of the binding in step (b) of the present invention can be referred to the above for the composition for detecting Salmonella enteritidis KCCM 12021, which is another embodiment of the present invention, and is omitted in order to avoid the excessive complexity described herein do.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides an oligopeptide that specifically binds to Salmonella enteritidis KCCM 12021.

(b) The present invention relates to a nucleic acid molecule which encodes the amino acid sequence of the above-mentioned oligopeptide, a recombinant expression vector comprising the nucleic acid molecule, a transformant which is transformed with the expression vector Provide sieve.

(c) The present invention provides a composition for detecting Salmonella enteritidis KCCM 12021 comprising the above-described oligopeptide and a detection kit.

(d) The present invention provides a method for detecting Salmonella enteritidis KCCM 12021 comprising the above-described oligopeptide.

(e) By using the present invention, it is possible to effectively and specifically detect Salmonella enteritidis KCCM 12021 in a short time by omitting the pretreatment such as sample extraction by recognizing the cell surface as it is.

(f) Using the present invention, it is possible to more easily detect Salmonella enteritidis KCCM 12021 by using an oligopeptide which is less immune than the antibody, structurally stable, chemically synthesized and economical.

FIG. 1 shows a plan view of M13 phage peptide screening for peptide-expressing phage screening that binds to cells. (a) immobilizing Salmonella enteritidis KCCM 12021 in each well of a PDL coated plate, (c) adding an M13 phage library expressing peptides composed of 12 random amino acids, (d) e) By combining the phage binding to Salmonella enteritidis KCCM 12021 by controlling various washing conditions and binding time, only a specific binding to Salmonella enteritidis KCCM 12021 is secured. (f) Finally, only the peptide expressing phage binding to Salmonella enteritidis KCCM 12021 is extracted. 1 (a) to 1 (e) are referred to as a round, and the above process is called biopanning.
Fig. 2 shows a 4-round bio-panning test condition for screening peptide expression phage binding to Salmonella enteritidis KCCM 12021. Fig. Salmonella typhimurium KCCM 11806 In order to detect a phage coding for a peptide having high binding specificity and specificity, the concentration of NaCl and Tween-20 per round was increased and the phage binding time was shortened, A phage with high binding specificity and specificity was detected in Uremium KCCM 11806.
FIG. 3 shows a table for the detection frequency, sequence results, and binding strength of phage of a phage encoding a peptide binding to Salmonella enteritidis KCCM 12021 according to bio-panning. A total of 60 phage plaques were obtained to bind to Salmonella enteritidis KCCM 12021. Forty-eight phage plaques were analyzed as coding for one type of peptide. One phage was found to bind effectively to Salmonella enteritidis KCCM 12021 at the picomolar level.
FIG. 4 is a graph showing a result of analyzing binding force against phage binding to Salmonella enteritidis KCCM 12021. FIG. Peptide expression phage binding to Salmonella enteritidis KCCM 12021 was amplified and analyzed for binding capacity to Salmonella enteritidis KCCM 12021 for phage.
Figure 5 shows a graph analyzing the specificity of phage binding to Salmonella enteritidis KCCM 12021. In order to analyze the specificity of bacteriophage binding to Salmonella enteritidis KCCM 12021, the binding force at each concentration was compared and analyzed by treatment with Vibrio bacteria and bovine serum proteins, one of the causative bacteria of food poisoning.
FIG. 6 is a graph showing the oligopeptide sequence and the binding force binding to the synthesized Salmonella enteritidis KCCM 12021. (A) peptide sequence in bacteriophage bound to Salmonella enteritidis KCCM 12021 was analyzed and (b) FITC, a fluorescent substance, was attached to analyze the binding force between the peptide and the bacteria.
FIG. 7 is a graph comparing the binding force at various concentrations of Vibrio bacteria and bovine serum proteins, one of the causative agents of food poisoning, to analyze the specificity of SEBP peptide bound to Salmonella enteritidis KCCM 12021 .
Figure 8 shows a table on the outbreak statistics of food poisoning (source: food poisoning statistics system) by causal bacteria in 2002-2013.

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

Example 1: Screening of bacteriophage specifically binding to Salmonella enteritidis KCCM 12021 using Phage display method

A 96-well plate (SPR) coated with poly-D-lycine (PDL) was used for the phage display. Salmonella cells (Korean Microorganism Conservation Center) increased the efficiency of phage display by stably fixing (1 × 10 ⁸ cells) through ionic bonding of PDL amine group and negative charge on plate surface. This fixed cell was loaded with a random phage peptide library [phage display library PhD ^™ (Phage Display ^™), which was designed to have 2.7 billion different amino acid sequences through a random array of 12 amino acids fused to the M13 phage gp3 minor coat protein Peptide Library Kit, New England Biolabs) was designed to allow higher specificity and binding force analysis during screening. In order to screen phage peptides binding to Salmonella enteritidis KCCM 12021, peptides were prepared by immobilizing the cells on a plate surface, binding the cells with an M13 phage peptide library, and binding them with high affinity through various binding times and washing conditions Expression phage were selected. The screening schedule and reaction conditions in this regard are shown in Figures 1 and 2, respectively. FIG. 1 is a plan view of M13 phage peptide screening for cell-binding peptide expression phage screening. (d, e) after binding the cells to a phage library expressing a peptide library consisting of 12 amino acids on the surface of M13 phage, and (c) binding the cells to the surface of the PDL Corning 96- After washing under various conditions, (f) the final combined phage was eluted to obtain a phage. The thus obtained phage is infected with E. coli (ER2738) and amplified. The amplified phage is bound to the cells again, and then the amplified phage is repeated under conditions such as the intensity of the washing condition and the reaction time, . This sequence of processes is called biopanning. Figure 2 is a plot of the four-step bio-panning conditions for detecting phage expressing peptides with high specificity and binding ability to cells. In each step, the reaction condition was changed by various washing conditions and short reaction time. Bio - panning was performed to obtain a phage having a peptide binding to cells. The thus obtained phage was infected with E. coli ER2738 cells, and amplified in LB medium. Then, about 60 phage plaques were selected and M13 phage genomic DNA (Single Strand DNA) was isolated and purified to obtain respective gene sequences The amino acid sequences of the peptide fragments, which were expressed on the phage surface protein (gp3 minor coat protein) and were designed to bind to the cells, were identified. The results are shown in Fig.

Example 2: Binding force analysis of peptide expression phage binding to Salmonella enteritidis KCCM 12021

In order to analyze the screening ability of the phage binding, the phage expressing the peptide was first amplified and then the concentration of the phage solution was determined by titering. Then, the binding force of each peptide-expressing phage was calculated by measuring the amount of bound phage by putting the phage into the cell-bound plate by concentration.

Specifically, an enzyme-linked immunosorbent assay (ELISA) was performed using an antibody (Anti-M13 Monoclonal Antibody) that recognizes the surface protein of the phage after the phage was added to the cell-bound plate. An ELISA signal corresponding to the concentration was displayed and fitted to the saturation curve to determine the apparent Kd value.

In the experimental procedure, Salmonella enteritidis KCCM 12021 (1 × 10 ⁸ cells / 200 μl) was fixed on a PDL-coated plate, and the amplified phage was added. The plate was fixed for 2 hours and washed 5 times with 1 × TBST washing solution , The anti-M13 antibody was bound for 1 hour. Next, the antibody-bound protein was washed 10 times with the same washing solution, and then a secondary antibody (HRP-conjugated Polyclonal Antibody) was bound for 1 hour. TMB (3,3 ', 5,5'-tetramethylbenzidine) Minute reaction, and 1 M sulfuric acid was added to terminate the reaction. The Kd value was determined by measuring at 450 nm. As a result, as shown in FIG. 3 and FIG. 4, the peptides expressing two peptides showed the binding power of picomole (pM) concentration, which shows excellent binding force and has a binding force corresponding to the binding force of the existing antibody- I could confirm.

Example 3: Bacteriophage specificity assay binding to Salmonella enteritidis KCCM 12021

In order to analyze the specificity of Bacillus thuringiensis KCCM 12021 binding to Salmonella enteritidis KCCM 12021, we analyzed the binding force at each concentration by treatment with Vibrio bacteria and bovine serum proteins, Gram negative bacteria, The specificity of bacteriophage binding to ritidis KCCM 12021 was analyzed. The measurement method is the same as the above method of binding force analysis. As a result, it was confirmed that the bacteriophage binding to Salmonella enteritidis KCCM 12021 specifically binds to Salmonella enteritidis KCCM 12021 by measuring the binding ability to Vibrio bovis and bovine serum proteins to a markedly low level (see FIG. 5).

Example 4 Peptide Sequence and Binding Assay Binding to Salmonella enteritidis KCCM 12021

In a peptide expression phage, a peptide (Salmonella Enteritidis Binding Phage, SEBP) sequence was analyzed from one bacteriophage specifically recognizing Salmonella enteritidis KCCM 12021, and the peptide sequence was combined with a fluorescent substance (Fluorescein isothiocyanate, FITC) (See Fig. 6 (a)).

The sequence is as follows.

SEBP # 1: Ac- QSHDQNNYNQRS GCGK (FITC) -NH2

The indicated amino acid sequence represents from the amino terminal (N-terminal). The amino acid K (Lysine) was added to the C-terminus to attach the FITC to the carboxy terminal, while the N-terminal was acetylated to remove the reactivity of the amino terminal. GCGK is added near the carboxy terminus of each amino acid sequence, which is added for application in the process to maximize the sensitivity of the fluorescent peptide probe STBP-GCGK (FITC). For analysis of the binding force of the peptides on Salmonella enteritidis KCCM 12021 bacteria, each of the peptides was diluted by concentration to a fixed PDL coating plate fixed with Salmonella enteritidis KCCM 12021. Peptides bound to bacteria in each well of plaques were measured for fluorescence intensity for each peptide concentration at the maximum wavelength of FITC (Excitation: 495 nm, emission: 520 nm) connected to the peptide using a fluorometer. The fluorescence signal corresponding to the concentration of the peptide was labeled and the Kd value was determined according to the saturation curve (Fig. 6 (b)).

Example 5: Peptide specificity assay binding to Salmonella enteritidis KCCM 12021

In order to analyze the specificity of the synthesized peptides binding to Salmonella enteritidis KCCM 12021, the binding affinity at each concentration was analyzed by comparing with the treatment of Vibrio bovis and Bovine serum proteins, one of the food poisoning causative bacteria, The specificity for each peptide binding to ritidis KCCM 12021 was analyzed. The measurement method is the same as the above method of binding force analysis. As a result of the analysis, it was confirmed that the peptide binding to Salmonella enteritidis KCCM 12021 specifically binds to Salmonella enteritidis KCCM 12021 by measuring the binding force to the Vibrio bovis and the bovine serum protein to be significantly low.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Peptide Probe for Detection of Salmonella Enteritidis <130> PN140548 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> Peptide probe for Salmonella Enteritidis (SEBP) <400> 1 Gln Ser His Asp Gln Asn Asn Tyr Asn Gln Arg Ser   1 5 10

Claims (8)

An oligopeptide that specifically binds to Salmonella enteritidis KCCM 12021 having the amino acid sequence of SEQ ID NO: 1.
A nucleic acid molecule characterized by encoding the amino acid sequence of the oligopeptide of claim 1.
A recombinant expression vector comprising the nucleic acid molecule of claim 2.
A transformant transformed with the expression vector of claim 3.
A composition for detecting Salmonella enteritidis KCCM 12021 comprising the oligopeptide of claim 1.
6. The composition according to claim 5, wherein the oligopeptide is labeled with one selected from the group consisting of chromogenic enzymes, radioactive isotopes, chromophores, luminescent materials and fluorescent materials.
A kit for detecting Salmonella enteritidis KCCM 12021 comprising the oligopeptide of claim 1.
Salmonella enteritidis KCCM 12021 detection method comprising the steps of:
(a) contacting the oligopeptide of claim 1 with a sample to be detected; And
(b) confirming whether or not the oligopeptide binds to Salmonella enteritidis KCCM 12021 that can be contained in the detection target sample.

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