KR102105157B1 - Peptide cleaved specifically by pepsin and kit for diasnosing Laryngopharyngeal Reflux comprising the same - Google Patents

Abstract

The present invention relates to a peptide specifically degraded by pepsin, a kit for diagnosing reflux pharyngitis comprising the same, and a method for diagnosing reflux pharyngitis using the same.

Description

Peptide cleaved specifically by pepsin and kit for diasnosing Laryngopharyngeal Reflux comprising the same}

The present invention relates to a peptide specifically degraded by pepsin, a kit for diagnosing reflux pharyngitis comprising the same, and a method for diagnosing reflux pharyngitis using the same.

Reflux pharyngitis refers to chronic inflammation of the larynx and pharynx or damage to the mucous membrane caused by the reflux of the contents, including gastric acid, through the esophagus to the larynx and pharynx. Although the number of patients complaining of reflux pharyngitis is gradually increasing as modern people's dietary habits change, industrialization, and urbanization have increased climate and atmospheric changes, there is still no simple and accurate way to diagnose them. Most of the treatment methods remain in the suppression of gastric acid secretion, so it is difficult to efficiently treat the symptoms, such as persisting symptoms even after treatment. Therefore, proper treatment through accurate and rapid diagnosis is essential.

The 24-hour esophageal acidity test, known as the gold standard for reflux pharyngitis, is costly, has a long test time, is inconvenient and difficult to use, and cannot predict the treatment response in patients with atypical symptoms. Patients with reflux pharyngitis often complain of non-specific symptoms, such as a sore throat, cough, and negative changes in the neck, rather than reflux symptoms, but it is difficult to accurately diagnose it, and many patients complaining of these symptoms can perform it immediately in the outpatient field and check the results. There is a need to develop a non-invasive and objective diagnostic method.

On the other hand, since pepsin is a proteolytic enzyme produced only in the stomach, pepsin detection technology in saliva can be useful as a sensitive and non-invasive method for reflux of gastric contents. Saritas et al. Demonstrated that detection of pepsin in saliva using a rapid lateral flow device is helpful in the diagnosis of reflux esophagitis (Saritas Yuksel, E. et al., Laryngoscope, 2012, 122 (6 ): 1312-1316), Darija et al. Found that the amount of pepsin in saliva was significantly higher in patients with reflux pharyngitis than in normal subjects (Darija, B. et al., Collegium Antropologicum, 2012, Supp. 2, 36: 83- 86). Accordingly, in the United States and other countries, simpler, more reliable, and non-invasive diagnostic methods are being developed.

In general, an immunochromatographic analysis method known as a rapid test method is a method for quantitatively and quantitatively analyzing a small amount of analyte in a short time using an antigen-antibody reaction, and diagnosis of various diseases Or, it is used in various fields such as medicine, agriculture, animal husbandry, food, military, and environment, including inspection. In the immunochromatographic analysis, an assay strip containing a reactant capable of reacting with an analyte to be detected and showing a change, or a device-type analytical device in which the analytical strip is mounted on a plastic case is generally used. . 1 is a cross-sectional view of an analytical strip used in conventional immunochromatographic analysis. As shown in FIG. 1, a typical analytical strip is a conjugate that conjugates a sample pad containing a liquid sample, a label that generates a detectable signal using a naked eye or a sensor, to a ligand such as an antigen or an antibody. Consists of a conjugate pad containing, an analyte in a sample and / or a porous membrane pad immobilizing a binding agent (antibody or antigen) specifically binding to the conjugate, and a moisture absorbing pad finally receiving a liquid sample, and these functional pads In the order listed above, they are connected in some overlapping form and attached on a solid support to be arranged continuously. When the analytical strip is mounted and used inside the plastic case, the sample inlet for dropping the sample at the position of the sample pad is located at the top of the case, and the result for confirming the test result is confirmed at the position where the binder of the porous membrane pad is fixed. A window is formed. As described above, in the immunochromatographic analysis method using an analytical strip, when a liquid sample is dropped on the sample pad, the liquid sample moves through the conjugate pad and the porous membrane pad by capillary action, and finally is accommodated in the moisture absorption pad. At this time, the conjugate contained in the conjugate pad also moves with the liquid sample, and when a substance to be analyzed exists in the sample, the conjugate mediates the analyte and binds with a binder fixed to the porous membrane pad (normally, "sandwich"). (Sandwich reaction).) As the conjugate and analyte competitively bind to the binding agent (commonly referred to as a "competition reaction"), the presence or absence of the analyte in the sample is visually or using a sensor. Can be detected. In order to use a commonly used sandwich reaction, two types of antibodies specific to the analyte are required, and the antibodies must react to different antigen binding sites of the same antigen. When using such an antibody, there is an advantage of selective and sensitive detection using the high specificity and affinity of the antibody, but due to the manufacturing process using animals or cells, it is difficult to produce, takes a long time, and consumes a lot of cost. It is not easy to obtain high-quality antibodies with high binding capacity to the desired antigen because the activity may be different depending on the period. In addition, since the antibody has low stability, it is affected by the environment such as pH and temperature, and thus, there is a problem that storage is difficult.

Accordingly, the present inventors, as a result of earnest research efforts to provide a kit for diagnosing reflux pharyngitis using saliva, a non-invasively collectable sample that contains an antibody for economical and easy storage, as a result of diagnosing reflux pharyngitis Using the proteolytic action of pepsin in saliva, an index that can be used, an amino acid sequence that can be specifically degraded only by pepsin and not degraded by other enzymes was designed. In addition, by using an appropriate combination of ligands, it is confirmed that a kit capable of diagnosing reflux pharyngitis, based on this, as well as qualitative / quantitative analysis of pepsin, which applies the principle of immunochromatography but does not contain antibodies, The present invention has been completed.

One object of the present invention is a peptide that is specifically degraded by pepsin, which has a labeling substance and a first ligand at one end, and a second ligand at the other end; And a third line specifically binding to the first ligand, a test line formed to have a predetermined width in a direction perpendicular to the flow of the fluid, and a fourth specifically binding to the second ligand. It is composed of a membrane pad including a control line formed in parallel to be spaced apart from the inspection line by a predetermined distance, including a ligand, and a moisture absorption pad that finally receives a liquid sample, and the membrane. A kit for diagnosing reflux pharyngitis, comprising a strip for immunochromatography in which a pad and a hygroscopic pad are partially overlapped, wherein the first and second ligands are different from each other, and the first and fourth ligands are different from each other. It is to provide a kit that is selected so that the 4th and 4th ligands and the 2nd and 3rd ligands are not combined with each other.

Another object of the present invention is the first step of deploying saliva collected from an individual suspected of developing reflux pharyngitis into the membrane pad of the kit for diagnosing reflux pharyngitis, and developing in the direction of the hygroscopic pad to cross the test line and the control line; A second step of identifying a signal by a labeling substance at the inspection line and the control line; And it provides a method for providing information for diagnosing reflux pharyngitis, comprising a third step of determining positive for a reflux pharyngitis when the signal is detected at the control line and the control line, and negative when the signal is detected only at the control line. .

Another object of the present invention is that two hydrophobic amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan are consecutively located, and 2 to each of the sock ends of the consecutively located hydrophobic amino acids A first step of preparing a series of peptides to which 10 amino acids have been added; And a second step of contacting a series of peptides prepared from the first step with pepsin, trypsin, amylase, lysozyme or mucin, and confirming whether or not the enzyme is decomposed by each enzyme. It is to provide a manufacturing method.

Another object of the present invention is to provide a peptide having an amino acid sequence of GDFLMGRDMRK (SEQ ID NO: 1), GDFLGGRDARK (SEQ ID NO: 2) or CGDFLMGRDMRK (SEQ ID NO: 3).

In order to achieve the above object, the first aspect of the present invention

A peptide specifically degraded by pepsin, having a labeling substance and a first ligand at one end and a second ligand at the other end; And

A test line formed to have a predetermined width in a direction perpendicular to the flow of the fluid, including a third ligand specifically binding to the first ligand, and a fourth ligand specifically binding to the second ligand It comprises a membrane pad (membrane pad) including a control line (control line) formed parallel to be spaced apart by a predetermined distance, including the inspection line, and the absorption pad (absorption pad) to finally receive the liquid sample, the membrane pad And a reflux pharyngitis diagnostic kit comprising a strip for immunochromatography in which the hygroscopic pads are connected in some overlapping form,

The first and second ligands, and the third and fourth ligands are different from each other, and the first and fourth ligands, and the second and third ligands are selected not to be bonded to each other.

For example, the immunochromatography strip included in the kit of the present invention includes a sample pad for accommodating a liquid sample and / or a reaction pad for reacting the sample with a peptide specifically degraded by pepsin. ). These pads may optionally be located at the front end of the membrane pad, i.e., on the other side of the moisture absorption pad. These pads are located in the order of the sample pad, the reaction pad, the membrane pad, and the moisture absorption pad, and may be arranged so that the sample on the solution may sequentially move toward the moisture absorption pad by partially overlapping through the end.

On the other hand, peptides that are specifically degraded by the pepsin included in the kit of the present invention are provided independently of the immunochromatography strip, and can be applied to the immunochromatography strip by reacting with a sample from the outside during diagnosis using this. . Alternatively, the immunochromatography strip may be provided in a bound or adsorbed state on a reaction pad included therein to react with pepsin in the sample on the reaction pad when the sample is developed. However, this is only an example of how to implement the kit, and the scope of the present invention is not limited thereto.

The present invention applies the principle of immunochromatography, so that a diagnostic kit that does not contain an antibody can be constructed using the specific peptide resolution of pepsin, i) not degraded by other enzymes in saliva, and specific by pepsin It is characterized by excavating a peptide having a specific amino acid sequence of a suitable length, which can decompose into ii) and exhibit water solubility to be moved through the analytical strip by reaction and capillary action with a solution merchant sample. Furthermore, by introducing a labeling substance at one end of the amino acid sequence, and introducing a different ligand to each of the two fragments that have been degraded, the fragments to which the labeling substance is bound are on the test line, and the other fragments that have been degraded and the peptides that have not been decomposed are on the control line. It can be used to diagnose reflux pharyngitis by forming a test line and a control line with a ligand corresponding to each to be bound.

The immunochromatography used in the conventional rapid test is an analysis method combining an immunoreaction principle based on an antigen-antibody reaction and a chromatography principle in which samples and reagents move along a medium by a mobile phase. The present invention uses a chromatographic principle in which double samples and reagents move along a medium by a mobile phase, but is specific in place of the antigen-antibody reaction in order to avoid the use of an antibody that is difficult to produce and easily denatured and has long-term storage. It is designed to take advantage of the binding between ligands. Specifically, the present invention utilizes the protein resolution of pepsin in detecting pepsin in saliva as a method for diagnosing reflux pharyngitis. For example, in resolving a protein, pepsin preferentially breaks the bonds between consecutive hydrophobic peptides, rather than cutting at random positions in the amino acid sequence. Thus, the present inventors discovered a peptide composed of an amino acid sequence that is specifically degraded by pepsin compared to other proteolytic enzymes or enzymes in saliva. In addition, as described above, in order to detect this phenomenon by specific binding between ligands, the present invention introduces different ligands into two fragments formed by cleavage of the peptide, respectively, and specific to each of these ligands. It is characterized by constructing a kit so that it is possible to confirm whether the peptide is degraded by forming a test line and a control line by using the ligands that bind to each other.

The term "labeled material" of the present invention means a material that generates a signal that can be detected with the naked eye or using a sensor. The labeling material may include latex particles, gold particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers, or magnetic particles, but is not limited thereto. In addition, the signal may be generated by itself due to the inherent characteristics of the labeling material, such as light emission, or may be generated by an external stimulus such as fluorescence.

The term "ligand" of the present invention means a substance that specifically binds to each other. For example, antibodies that specifically bind to an antigen, ligands that specifically bind to a specific receptor, DNA pairs of complementary sequences that hybridize specifically to each other, and the like act as ligands with each other. In addition, in the present invention, the ligand may be used without limitation as long as it is a substance exhibiting the properties defined above. As used herein, the meaning of a ligand means a substance that specifically binds to each other as defined above, unless specifically indicated as a ligand specifically binding to a specific receptor. For example, the first ligand may be a maleimide group, and the third ligand may be a material containing a thiol group specifically binding thereto, for example, biotin containing cysteine having a thiol group, but is not limited thereto. For example, the second ligand may be biotin, and the fourth ligand may be streptavidin specifically binding thereto, but is not limited thereto. As another example of the first and third ligands, and the second and fourth ligands, a material capable of selectively bonding histidine and nickel may be used, but is not limited thereto. Other examples of the first ligand and the third ligand pair that specifically bind to the first ligand may be a single-stranded DNA pair capable of hybridizing by complementary binding to each other to form double-stranded DNA, but are not limited thereto.

The test line selectively captures a fragment of the first ligand attached to the labeling substance formed by the peptide contained in the sample pad degraded by pepsin and the presence of pepsin in the sample. It is formed in order to be configured to include a third ligand that specifically binds to the first ligand for such selective capture.

On the other hand, the control line is formed to capture the other fragment that does not contain a labeling substance among the degraded peptide fragments after the reaction of the peptide contained in the sample pad with pepsin and / or unreacted peptides, that is, undigested peptide. , For this capture, a second ligand is bound to the other end of the peptide-bound material, and the control line may be configured to include a fourth ligand capable of specifically binding thereto. The control line may be a standard for determining whether the kit is operating normally. The specific operating principle will be described later.

The peptide specifically degraded by the pepsin may include two hydrophobic amino acids located in series, selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan. The two amino acids may be the same or different from each other. The cleavage site for pepsin can be provided by including a portion composed of the two hydrophobic amino acids. Furthermore, for development into a solution phase, polar and / or positive or negatively charged amino acids may be further included, and the peptide may be, for example, 5 to 20 amino acids in total, specifically, 10 to 15 amino acids. It may be composed of amino acids, but is not limited thereto. For example, the peptide specifically degraded by the pepsin, the amino acid sequence of SEQ ID NO: 1, that includes -FL- as a cleavage site by pepsin, that is, GD FL MGRDMRK, the amino acid sequence of SEQ ID NO: 2, that is, GD FL GGRDARK Or, it may be a peptide of the amino acid sequence of SEQ ID NO: 3, that is, the sequence represented by CGD FL MGRDMRK.

For example, the diagnostic kit of the present invention may further include a sample pad and / or a reaction pad, as described above. In this case, when the peptide is included in the reaction pad, a flow control separation membrane for delaying the flow of the sample, a heating plate for improving the rate of decomposition reaction by pepsin, or both, is attached to the reaction pad so that the peptide can sufficiently react with the sample. It may further include, but is not limited thereto.

Specifically, the flow control separation membrane is a typical example in which a material such as vinyl is added between the membranes, thereby suppressing or blocking the flow of a liquid sample and allowing it to flow back through the membrane upon removal, in addition to hydrophobicity or superhydrophobicity. It may also be implemented by controlling the flow of the liquid sample on the membrane through a surface treatment by interfering with or delaying the flow, or by additionally configuring a channel through which the fluid flows on the membrane to control the width or number of channels.

Meanwhile, the heating plate may constitute a heating plate using a chemical reaction in consideration of manufacturing cost. At this time, it can be implemented using a chemical used in a hand stove, for example, sodium acetate or sodium thiosulfate and a metal plate. As another example, an inexpensive electric heating film may be provided, which may be implemented using a graphene heating film having uniform heating characteristics, but is not limited thereto.

Both of these flow control separators and heating plates are provided at the bottom of the reaction pad to control the reaction time and / or reaction temperature to promote the peptide decomposition reaction by pepsin, thereby helping to achieve efficient reaction and / or sensitive detection.

The liquid sample used in the diagnostic kit of the present invention may contain saliva. For example, the liquid sample may be a solution in which saliva is diluted in a buffer solution to adjust the concentration and / or viscosity and impart fluidity, but is not limited thereto.

As described above, the diagnostic kit of the present invention uses the principle of chromatography in which a mobile phase containing an analyte moves along a medium. Therefore, for analysis using this, a mobile phase is required to move the sample containing the analyte along the strip. The buffer not only acts as a mobile phase to move the sample along the analytical strip, but can also serve as a diluent to dilute the sample if necessary. For example, as the buffer solution, a conventional buffer solution such as a phosphate buffered solution (PBS) having a concentration of 10 mM to 1 M, a nonionic or amphoteric surfactant, methanol, or a mixture thereof can be used without limitation. In addition, a pH adjusting agent or citric acid may be further included to provide conditions suitable for the decomposition reaction of pepsin. Specifically, a mixed solution of methanol and 0.1 M citric acid solution in 5% by volume and 95% by volume, respectively, may be used, but is not limited thereto.

Thus, the diagnostic kit of the present invention may be provided with a buffer as a mobile phase for dilution of samples and / or development on a strip, but is not limited thereto.

The diagnostic kit of the present invention is provided with a moisture absorption pad, and the sample is transferred from a sample pad into which a liquid sample is introduced, a reaction pad containing a peptide specifically degraded by pepsin, and a sample is transferred to a membrane pad equipped with a test line and a control line. It can provide the driving force to be.

For the development of such a sample, the series of pads were formed using a membrane pad so that they could be moved by capillary action, and one end thereof was equipped with a moisture absorbing pad to provide a driving force for transporting the sample. The moisture absorption pad may be partially overlapped with the membrane pad. As the membrane, a nitrocellulose membrane, a glass fiber membrane, a polyethersulfone (PES) membrane, a cellulose membrane, a nylon membrane, or a combination thereof may be used. It is not limited to this.

The diagnostic kit may additionally include a solid support on the bottom. As the solid support, a plastic support can be used, and thus, by attaching the kit on the solid support, the durability can be increased, and handling and storage can be facilitated. In addition, it is possible to facilitate the mounting of an additional outer case. As a plastic material that can be used as the solid support, a polypropylene film, a polyester film, a polycarbonate film, an acrylic film, etc. may be used, but is not limited thereto.

The diagnostic kit can be additionally fixed in the case. The inside of the lower case may be provided with a plurality of guides and / or kit supports for arranging and fixing or compressing the diagnostic kit in an appropriate position. Optionally, the guide and the kit support may be provided in the upper case at positions corresponding to the guide and kit support provided in the lower case. That is, the guide and / or kit support portion may be formed in the lower case or the upper case and the lower case as needed. In addition, the upper case may be provided with a result confirmation window for detecting a signal from the labeling substance at a position corresponding to the sample input port and the inspection line. The sample inlet is formed in the form of a hole or a slit at a point sufficiently spaced so that the test line and the sample are developed along the membrane pad at one end of the membrane pad, that is, on the opposite end where the moisture absorption pad is located relative to the test line. You can. The result confirmation window may be formed in a size large enough to be discernable from the outside to the naked eye or through a sensor, including a control line on the membrane pad and / or an additional control line in some cases. . As long as the inspection line and / or the control line can be identified, the size and shape can be formed without limitation.

The upper and lower cases may be manufactured using a conventional plastic material, for example, polycarbonate, acrylonitrile butadiene styrene (ABS), and the like may be used, but is not limited thereto. The upper and lower cases may be separately manufactured and provided with engaging grooves, engaging projections, or the like, and combined with conventional means, and in some cases, may be integrally manufactured.

The second aspect of the present invention includes a first step of introducing saliva collected from an individual suspected of developing reflux pharyngitis into the membrane pad of the kit of the first aspect and developing it in the direction of the hygroscopic pad to cross the test line and the control line; A second step of identifying a signal by a labeling substance at the inspection line and the control line; And it provides a method for providing information for diagnosing reflux pharyngitis, comprising a third step of determining positive for a reflux pharyngitis when the signal is detected at the control line and the control line, and negative when the signal is detected only at the control line.

For example, in the diagnostic kit of the present invention, the control line is included in the reaction pad, and when the liquid sample is added, the labeling substance of the peptide that is specifically degraded by the pepsin moving toward the hygroscopic pad according to the fluid sample is bound to the other end that is not bound Including a fourth ligand that specifically binds to the second ligand, which is spaced apart by a certain distance from the test line and formed in parallel, wherein the peptide fragment and the unresolved peptide bound by the pepsin are bound to the second ligand. As a result, as long as the moisture-absorbing pad contains a sufficient amount of peptide, the control line emits a signal of the labeling substance by binding of the unresolved peptide. Therefore, if a signal is not detected from the control line in the third step, it may be determined that the diagnosis is invalid. This may occur when the kit itself is defective, for example, if the amount of peptide contained in the hygroscopic pad is not sufficient, the flow of the liquid sample is not smooth, or the pepsin concentration in the sample is too high.

In the third aspect of the present invention, two hydrophobic amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan are consecutively located, and 2 to 10 are respectively placed on the sock ends of the continuously located hydrophobic amino acids. A first step of preparing a series of peptides to which two amino acids have been added; And a second step of contacting a series of peptides prepared from the first step with pepsin, trypsin, amylase, lysozyme or mucin, and confirming whether or not the enzyme is decomposed by each enzyme. It provides a method of manufacturing.

In order to easily confirm whether or not the enzyme is decomposed in the second step, a labeling substance may be introduced at one end of the peptide. On the other hand, in order to facilitate separation of the cut two fragments, the peptide can be bound to a predetermined particle, and the two cut fragments are separated from each other by a simple method of centrifugation using the weight difference by the particles. Can be separated. At this time, by designing the labeling material and the particles to be located in different fragments, the signal of the labeling substance is measured for one recovered fragment, and the changes are observed to detect whether the peptide is cleaved by each enzyme and / or You can check the degree. However, this is only one example for performing the second step, and the scope of the present invention is not limited thereto, and the second step may be performed by applying various methods known in the art.

For example, the peptide may be a peptide having an amino acid sequence designed to be soluble in water for development into a solution phase. Specifically, polar and / or positive or negatively charged amino acids may be additionally included in positions other than the two consecutively located hydrophobic amino acids that provide a cleavage site by pepsin, and the peptide may be, for example, 5 in total. It may be composed of 20 to 20 amino acids, specifically, 10 to 15 amino acids, but is not limited thereto.

Furthermore, the peptide may be used in a kit for diagnosing reflux pharyngitis. To this end, an appropriate labeling material and / or one or more ligands may be introduced between the sock end and sequence of the peptide.

The fourth aspect of the present invention provides a peptide having an amino acid sequence of GD FL MGRDMRK (SEQ ID NO: 1), GD FL GGRDARK (SEQ ID NO: 2) or CGD FL MGRDMRK (SEQ ID NO: 3).

As described above, the peptide of the present invention has a sequence designed to be specifically degraded by pepsin, which is degraded only by pepsin, and is a protease, trypsin, or an enzyme present in saliva, such as amylase, lysozyme, or mucin. Does not decompose. This suggests that by using the peptide of the sequence, pepsin can be qualitatively and / or quantitatively detected without being influenced by other enzymes contained in saliva used as a sample.

Specifically, the peptide of the present invention is degraded by pepsin, GD (SEQ ID NO: 4) and MGRDMRK (SEQ ID NO: 5), GD (SEQ ID NO: 4) and GGRDARK (SEQ ID NO: 6), or CGD (SEQ ID NO: 7) and MGRDMRK (SEQ ID NO: 5) can be cut into two fragments.

The peptide of the novel amino acid sequence of the present invention is hardly degraded by other proteolytic enzymes or enzymes in saliva, and is specifically degraded only by pepsin, and thus can be useful in the diagnosis of reflux pharyngitis. On the other hand, by constructing a diagnostic kit using a peptide instead of an antibody by using the specific decomposition of the peptide by pepsin, an kit for diagnosing reflux pharyngitis that is economical and advantageous for long-term storage can be provided.

1 is a view schematically showing a cross-section of an analytical strip used in conventional immunochromatographic analysis.
Figure 2 is a schematic diagram showing the principle of operation of the peptide-based reflux pharyngitis diagnostic strip according to the present invention.
3 is a diagram showing a peptide of an amino acid sequence that is specifically degraded by pepsin according to the present invention, a principle of pepsin detection using the same, and a decomposition rate according to reaction time.
4 is a view showing the resolution according to the pepsin concentration for the amino acid sequence that is specifically degraded pepsin according to the present invention.
5 is a diagram showing the specific degradation of pepsin compared to other enzymes of the amino acid sequence according to the present invention.
Figure 6 (A) for the detection of pepsin containing oligonucleotides for selective capture of peptides of amino acid sequences that are specifically degraded by pepsin according to the present invention, gold nanoparticles and peptide fragments degraded by gold pepsin as colorimetric markers It is a diagram showing the pepsin-specific resolution of the complex and (B) the complex.
FIG. 7 shows the structure of (A) maleimide-functionalized pepsin-specific peptide for selectively detecting specific fragments when the peptide specifically degraded by pepsin according to the present invention is degraded by pepsin, (B) of the peptide It is a diagram showing the optimization of the specific binding force according to the functionalization and (C) the pH for the selective binding.
8 is a diagram showing the detection of pepsin by an assay strip using immunochromatography according to the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example  One: Peptide  Synthesis and separation

In order to synthesize the desired peptide of any sequence, it was performed according to a general peptide solid-phase synthesis method (see Wang C. Chan and Peter D. White, Fmoc Solid phase peptide synthesis, Oxiford). Specifically, by using an automatic synthesizer (ASP48S, Peptron, Inc.), Fmoc-SPPS (9-Fluorenyl methyl oxycarbonyl solid phase peptide synthesis) method was used to couple amino acid monomers from the C-terminus one by one. All monomer raw materials used for peptide synthesis are protected with Fmoc at the N-terminus, and residues are amino acids protected with trityl (Trt), t-butyloxycarbonyl (Boc), t-butyl (t-Bu), etc. Did. As a coupling agent, HBTU (2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate / HOBt (hydroxybenzotriazole) / NMM (N-methylmorpholine) was used.

The protected amino acid (8 equivalents) and the coupling agent HBTU (8 equivalents) / NMM (16 equivalents) were dissolved in DMF (dimethyl formamide) and added, followed by reaction at room temperature for 2 hours. Then, in order to remove Fmoc, 20% (v / v) piperidine / DMF was added and reacted twice at room temperature for 5 minutes. The above two reactions were repeated to prepare a peptide having a desired sequence.

Subsequently, the separation of the peptide from the resin and the amino acid protecting group is trifluoroacetic acid (TFA) / EDT (1,2-ethanedithol) / thioanisole / TIS (triisopropylsilane) / H ₂ O (90 / 2.5 / 2.5 / 2.5 by weight). /2.5). The prepared mixed solution was treated with an excessive amount of the diethyl ether solvent stored in the refrigerator to form a precipitate, and the resulting precipitate was completely precipitated by centrifugation, and then excess TFA, EDT, thioanisole and TIS were first removed. . The same process was repeated twice or so to obtain a solidified precipitate.

The obtained precipitate was water containing 0.1% (v / v) TFA using a high performance liquid chromatography apparatus (Shimadzu Prominence HPLC, Japan) equipped with an Ace C18 column (250 mm × 22 mm, 10 μm, UK)- It was purified by separation using an acetonitrile linear gradient (acetonitrile concentration: 10 to 75% (v / v)). The molecular weight of the purified peptide was confirmed by LC / MS (Shimadzu LC / Mass-2020, Japan), and the purified fraction was lyophilized to obtain a white powder in the form of TFA salt.

Example  2: pepsin specific Peptide  Verification

In order to experimentally confirm pepsin-specific degradation of the peptide having the amino acid sequence of SEQ ID NO: 1 according to the present invention, 3 'terminal K is connected to biotin, 5' terminal G is a linker 6-aminohexanoic acid (6 -Aminohexanoic acid) was used to synthesize a peptide linked to FITC, a phosphor, and confirmed by mass spectrometry.

FITC- (6-aminohexanoic acid) -GDFLGRDMRK (biotin)

-Mass Analysis

Instrument: SHIMADZU LCMS-2020

MS Expected: 2053.5

MS Found: 2053

As prepared above, a peptide having an amino acid sequence of SEQ ID NO: 1, which is a fluorescent substance FITC labeled at one end and biotin-linked at the other end, was prepared at a concentration of 10 μM and reacted with pepsin at a concentration of 1,000 ng / mL. The reaction was performed at 42 ° C, and immediately after starting the reaction, 5 minutes, 10 minutes and 30 minutes later, biotin-bound peptide fragments were recovered using agarose beads with streptavidin bound to the surface. The fluorescence of FITC was measured from the recovered peptide fragment to quantify the amount of peptide cut by pepsin. Upon digestion with pepsin, the fragments labeled with the phosphor FITC were removed by washing, so it was possible to confirm the degree of degradation of the peptide with pepsin from the decrease in fluorescence. Fluorescence intensity according to the measured reaction time is shown in FIG. 3. As shown in Figure 3, it was confirmed that about 30% of the peptide was degraded within 5 minutes after the initiation of the reaction, and 70% or more after 30 minutes.

Example  3: according to pepsin concentration Peptide  Resolution

In order to confirm the possibility of quantitative analysis according to the concentration of pepsin, peptide degradation was measured in a similar manner to Example 2 while varying the concentration of pepsin. 5, 10, 20, and 40 μM concentrations of peptide solutions were prepared, respectively, and 250, 500, 1,000, and 2,000 ng / mL concentrations of pepsin solutions were reacted for 5 minutes at room temperature, and the amount of remaining peptides was calculated. It is shown in. After the peptide of 10 μM concentration, which showed linear change according to the pepsin concentration, was reacted with the pepsin solution of 0, 10, 50, 100, 500 and 1,000 ng / mL concentration for 30 minutes again, the amount of the remaining peptide was calculated. It is shown together in 4. As shown on the right side of FIG. 4, when a peptide having a concentration of 10 μM was reacted, it was confirmed that a linear decomposition rate according to the pepsin concentration ranged from 0 to 1,000 ng / mL. This indicates that quantitative analysis of pepsin concentration is possible by measuring a fluorescence signal or color change in a corresponding range.

Example  4: Depending on the type of enzyme Peptide  Decomposition rate

In order to confirm the possibility of cleavage of pepsin-specific peptides specifically designed according to the present invention by enzymes other than pepsin and other proteolytic enzymes, trypsin (1.5 units, instead of pepsin (1.5 units, 50 ng)) 1500 ng), amylase (1.5 units, 5000 ng), lysozyme (1.5 units, 150 ng) and mucin (1000 ng) were reacted for 5 minutes and 1 hour, respectively, and then in a similar manner to Example 1, fluorescence intensity was changed. Was measured, and the results are shown in FIG. 5. As shown in Figure 5, the agarose bead itself (resin) did not generate any fluorescence, and when treated with pepsin compared to the fluorescence signal of the peptide itself (PEP-Only) without any enzyme treatment, within 5 minutes Although the fluorescence intensity was reduced to less than 5% in the reaction of, when the other enzymes were treated, only a decrease within 15% within 5 minutes, and a decrease of up to 35% after 1 hour. This is because the peptide having the amino acid sequence of SEQ ID NO: 1 designed according to the present invention is a sequence that is specifically degraded by pepsin, using this sequence, it is possible to qualitatively and quantitatively detect pepsin in a sample without interference with other sequences. To indicate.

Example  5: degraded by pepsin Peptide  For selective binding of fragments Oligonucleotide -Preparation of peptide complex and evaluation of specific degradation performance by pepsin

As a method for selectively detecting fragments degraded by pepsin of a pepsin-specific peptide designed according to the present invention, a pepsin-specific peptide complex is synthesized in which an oligonucleotide is coupled to one end to utilize hybridization of complementary DNA It was prepared using a post-synthetic coupling method. As a pepsin-specific peptide, a peptide having the amino acid sequence of SEQ ID NO: 3 was used.

Step 1 : First, considering the secondary structure such as G / C content of the sequence, hairpin, self-dimer, and the like, and then pre-validating using OligoAnalyzer 3.1 program (IDT, Inc.), the result And oligonucleotides were prepared. The length of the oligonucleotide was set to 21mer, and a thiol group at the 5 'end and a maleimide group at the 3' end were introduced. The synthesized oligonucleotide having functional groups at both ends was purified by PAGE (polyacrylamide gel electrophoresis), and the degree of purification by MALDI-TOF QC was confirmed. The prepared oligonucleotide sequence is 5 '-[thioC6] -AAg gAg ggg gAg AAg TgA ggT- [maleimide] -3' (SEQ ID NO: 8), 5 'as a functional group for binding to a colorimetric gold nanoparticle It contains a thiol group at the terminal, and is designed to bind to the C terminal of the pepsin-specific peptide through a maleimide group at the 3 'terminal.

Step 2 : Next, in order to combine the oligonucleotide having a functional group at the sock end obtained from step 1 with a peptide having the amino acid sequence of SEQ ID NO: 3, neutral conditions of these oligonucleotides and peptides at a concentration of 1: 2 ( 100 mM KH2PO4, pH 7.2) for 4 hours or more at room temperature. The conjugate of the obtained oligonucleotide and peptide was electrophoresed on a 10% TBE-urea polyacrylamide gel, and the oligonucleotide was stained with methylene blue to confirm the position of the conjugation band on the gel. On the basis of this, the band was cut from the gel, and conjugation was extracted using the Crush and Soak technique. The concentration of the oligonucleotide-peptide conjugate extracted with nanodrops was calculated.

Step 3 : Finally, the disulfide bond (-SS-) contained in the 5 'end of the oligonucleotide in the oligonucleotide-peptide conjugate obtained from step 2 is reduced by reducing tris (2-chloroethyl) phosphate (TCEP) beads It was reduced to a thiol group (-SH) with bead) to bind gold nanoparticles as a colorimetric marker (Fig. 6 (A)).

In order to confirm whether the oligonucleotide-peptide complex specifically designed according to the present invention is specifically degraded by pepsin, 2,000 oligonucleotide-peptide complexes containing gold nanoparticles prepared according to steps 1 to 3 were introduced. It was reacted with the ng / mL concentration of pepsin. The reaction was performed at 42 ° C. for 30 minutes, and after completion of the reaction, biotin-bound peptide fragments were removed by centrifugation using streptavidin-coupled agarose beads on the surface. Thereafter, the absorbance of the supernatant was measured to quantify gold nanoparticles, thereby evaluating the decomposition performance by pepsin. Specifically, the oligonucleotide-peptide complex in which the gold nanoparticles are introduced includes gold nanoparticles at one end and biotin at the other end, and after reacting with pepsin, streptavidin-coupled agarose beads When centrifuged after contacting, the non-degraded oligonucleotide-peptide complex containing biotin at one end and the other fragment not containing gold nanoparticles among the complex degraded by pepsin are bound to the beads and precipitate, and the supernatant is Since only the fragment containing gold nanoparticles was included among the complexes degraded by pepsin, the degree of decomposition of the peptide by pepsin could be calculated by measuring the absorbance of the recovered supernatant to quantify the gold nanoparticles. That is, a high absorbance measured in the supernatant indicates that more peptides were decomposed by pepsin.

Example  6: As a ligand for realizing on-site diagnostic strip for pepsin detection Peptide  Experimental verification of functionalization and selective binding by the ligand

In order to implement a diagnostic strip that can be used for actual in situ diagnosis using the pepsin-specific peptide designed according to the present invention, the amino acid of SEQ ID NO: 2 so that a fragment containing a colorimetric marker of a peptide degraded by pepsin can be identified on a test line A maleimide group was introduced as a ligand on the side to which the colorimetric marker of the peptide having the sequence was bound. In addition, TAMRA, which can confirm the color change with the naked eye as well as fluorescence, was introduced as a colorimetric marker. The thus synthesized peptide was separately identified as follows:

TAMRA-MiniPEG2-K (4-maleimidobutyryl) -MiniPEG2-GDFLGGRDARK (biotin);

R _t = 5.40 min, various concentration gradients from 5% (v / v) to 100% (v / v) DW / acetonitrile with 0.01% (v / v) TFA over 20 min;

MS (ESI) 2412.96 m / e, [M + H] ⁺ = 2413.

Cysteine having a thiol group is included to confirm whether selective detection of a peptide fragment containing the same is possible by the maleimide when cleaved by pepsin using a peptide functionalized with maleimide synthesized and identified as described above. It was tested as follows using a biotin (CK-Biotin) solution. First, a peptide of 10 μM concentration was reacted with a 2,000 ng / mL pepsin solution at 42 ° C. for 30 minutes, and then the biotin-bound peptide fragment was removed. Subsequently, a biotin solution (2 mM CK-Biotin) containing cysteine having a thiol group was added to the supernatant (pH 3 to 4) containing a peptide fragment containing TAMRA and maleimide groups, and maintained at room temperature for 20 minutes. Was reacted to bind CK-Biotin through a maleimide group. After collecting the CK-Biotin bound to the maleimide group with agarose beads bound with streptavidin, the fluorescence of the remaining supernatant was measured to quantitatively analyze the binding of the maleimide group to CK-Biotin, and the results are shown. 7 (B). At this time, the structure of the pepsin-specific peptide functionalized with the maleimide used is schematically shown in FIG. 7 (A). As shown in FIG. 7 (B), little fluorescence was observed from the supernatant from which the peptide fragment bound to CK-Biotin was removed (about 6.2% of the measured fluorescence before CK-Biotin binding). This indicates that the peptide fragment degraded by pepsin effectively bound to CK-Biotin through a maleimide group functionalized thereon.

On the other hand, in order to minimize the measurement error caused by a false positive signal due to the binding of the functionalized peptide with CK-Biotin, which has not been degraded by pepsin, the functionalized peptide and CK-, which have not been degraded by pepsin, while changing reaction conditions The binding strength of biotin was confirmed. First, as described above, a peptide functionalized with maleimide at a concentration of 10 μM was reacted with agarose beads bound with streptavidin for 10 minutes at room temperature to bind. Thereafter, a CK-Biotin solution capable of binding to maleimide groups was added to further react. At this time, the pH of the reaction solution was adjusted to 2 (0.2 M citric acid solution), 4 (1 × PBX), and 7 (1 × PBX), respectively, and the reaction was performed at room temperature for 10 minutes, followed by streptavidin-bound agar. After collecting the loss beads, the fluorescence of the collected beads was measured to confirm the degree of binding, and the results are shown in FIG. 7 (C). As shown in FIG. 7 (C), the binding degree of maleimide group and CK-Biotin was different according to the pH, and the concentration of maleimide and CK-Biotin ranged from about 31% at pH 7 to about 7% at pH 2. It was found that the binding force, pH-dependently decreased. This indicates that it is possible to selectively detect only peptide fragments degraded by pepsin by performing a reaction with CK-Biotin at a low pH, and considering using a citric acid solution of pH 2 when collecting saliva as a sample for diagnosis, It suggests that the collected saliva can be directly used for diagnosis without any treatment.

Example  7: On-site diagnostic strip for pepsin detection

In order to confirm whether the presence or absence of pepsin in a sample can be detected using a diagnostic strip comprising functionalized pepsin-specific peptides functionalized in accordance with Example 6 of the present invention, having a thiol group on the nitrocellulose membrane Cysteine-containing biotin was fixed to the test line and streptavidin to the control line to briefly construct a pepsin detection strip.

First, a peptide having a amino acid sequence of SEQ ID NO: 2, which is a phosphor and a TAMRA labeled with a red color visually identifiable at one end, and a maleimide group is introduced at the other end, and biotin-linked at the other end, was prepared at a concentration of 2 mM. As a comparison group, the pepsin was flowed through the strip as it was not reacted. In the experimental group, the peptides modified at both ends as described above were reacted with pepsin at a concentration of 60 μg / mL, and then flowed to a new strip. The strips of the comparison and experimental groups were visually observed and the results are shown in FIGS. 6 (A) and (B), respectively. As shown in Figure 6, in the case of the control group that did not react with pepsin, only the control region turned red (Fig. 6 (A)), but in the case of the experimental group developed by reacting with pepsin, both the test line and the control line turned red. It was confirmed that it changed. This indicates that the pepsin-specific peptide designed according to the present invention can be used to detect the presence or absence of pepsin in a sample together with the strip of the above-described configuration.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Peptide cleaved specifically by pepsin and kit for diasnosing          Laryngopharyngeal Reflux comprising the same <130> KPA180523-KR <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Pepsin-specific peptide 1 <400> 1 Gly Asp Phe Leu Gly Arg Asp Met Arg Lys   1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Pepsin-specific peptide 2 <400> 2 Gly Asp Phe Leu Gly Gly Arg Asp Ala Arg Lys   1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Pepsin-specific peptide 3 <400> 3 Cys Gly Asp Phe Leu Met Gly Arg Asp Met Arg Lys   1 5 10 <210> 4 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 1st fragment of Pepsin-specific peptide 1 and 2 <400> 4 Gly Asp   One <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 2nd fragment of Pepsin-specific peptide 1 and 3 <400> 5 Met Gly Arg Asp Met Arg Lys   1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 2nd fragment of Pepsin-specific peptide 2 <400> 6 Gly Gly Arg Asp Ala Arg Lys   1 5 <210> 7 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 2nd fragment of Pepsin-specific peptide 3 <400> 7 Cys Gly Asp   One <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide for chimeric complex <400> 8 aaggaggggg agaagtgagg t 21

Claims (14)

A peptide specifically degraded by pepsin, having a labeling substance and a first ligand at one end and a second ligand at the other end; And
A test line formed to have a predetermined width in a direction perpendicular to the flow of the fluid, including a third ligand specifically binding to the first ligand, and a fourth ligand specifically binding to the second ligand It comprises a membrane pad (membrane pad) including a control line (control line) formed parallel to be spaced apart by a certain distance, including the inspection line, and a moisture absorption pad (absorption pad) to finally receive the liquid sample, the membrane pad And a reflux pharyngitis diagnostic kit comprising a strip for immunochromatography in which the hygroscopic pads are connected in some overlapping form,
The first and second ligands, and the third and fourth ligands are different from each other, and the first and fourth ligands and the second and third ligands are selected not to be bonded to each other,
The peptide that is specifically degraded by the pepsin has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
According to claim 1,
The peptide that is specifically degraded by the pepsin is a kit comprising two hydrophobic amino acids, consecutively located, selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan.
delete According to claim 1,
The immunochromatography strip is a kit that further includes a sample pad, a reaction pad, or both.
According to claim 4,
The reaction pad kit further comprises a flow control membrane, a heating plate or both.
According to claim 1,
The liquid sample is a kit that contains saliva.
Saliva collected from a subject suspected of developing reflux pharyngitis is introduced into the membrane pad of the kit of any one of claims 1, 2, and 4 to 6, and the hygroscopic pad passes through a test line and a control line. A first step of deploying in a direction;
A second step of identifying a signal by a labeling substance at the inspection line and the control line; And
A method for providing information for diagnosing reflux pharyngitis, comprising a third step of determining positive for a reflux pharyngitis when a signal is detected at the control line and the control line, and negative when the signal is detected only at the control line.
The method of claim 7,
In the third step, if a signal is not detected from the control line, it is determined that the diagnosis is not valid.
A series of two hydrophobic amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, and tryptophan, and a series of 2 to 10 amino acids each added to the socks ends of the consecutively located hydrophobic amino acids. A first step of preparing a peptide; And
A second step of contacting a series of peptides prepared from the first step with pepsin, trypsin, amylase, lysozyme or mucin, and confirming whether or not the enzyme is decomposed by each enzyme. As a manufacturing method,
The method of manufacturing a peptide specifically degraded by the pepsin has an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
The method of claim 9,
The peptide is a water-soluble manufacturing method.
The method of claim 9,
The peptide is a method of manufacturing that is used in a kit for diagnosing reflux pharyngitis.
A composition for detecting reflux pharyngitis comprising a peptide having an amino acid sequence of GDFLMGRDMRK (SEQ ID NO: 1), GDFLGGRDARK (SEQ ID NO: 2) or CGDFLMGRDMRK (SEQ ID NO: 3).
The method of claim 12,
The composition is not degraded by the protease, trypsin, or the enzymes present in saliva, amylase, lysozyme or mucin, but is specifically degraded by pepsin.
The method of claim 12,
Two fragments of GD (SEQ ID NO: 4) and MGRDMRK (SEQ ID NO: 5), GD (SEQ ID NO: 4) and GGRDARK (SEQ ID NO: 6), or CGD (SEQ ID NO: 7) and MGRDMRK (SEQ ID NO: 5), respectively, by pepsin. The composition will be cut into.

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