JP6587133B2 - Microbial binding peptides and uses thereof - Google Patents

Description

  The present invention relates to a microorganism-binding peptide and use thereof.

  There are various pathogenic bacteria around us, and there is a risk of developing various diseases due to the pathogenic bacteria. For example, Escherichia coli and Salmonella are known to cause diarrhea and abdominal pain in addition to tuberculosis caused by tuberculosis and cholera caused by cholera. In order to prevent such infectious diseases, development of simple detection systems for various pathogenic bacteria is required regardless of developed countries or developing countries.

  By the way, in animal cells including humans, it is known that an immune defense mechanism works when Toll-like receptors recognize pathogenic microorganisms. One of Toll-like receptors, Toll-Like Receptor 4 (TLR4), is known to recognize endotoxin lipopolysaccharide (LPS) in the cell wall of Gram-negative bacteria (for example, see Non-Patent Document 1). reference.). In addition, Toll-Like Receptor 5 (TLR5), which is one of Toll-like receptors, is known to recognize flagellin, which is a flagellar constituent protein of bacteria (see, for example, Non-Patent Document 2).

Park B.S. and Lee J.O., Recognition of lipopolysaccharide pattern by TLR4 complexes., Exp. Mol. Med., 45 (12), e66, 2013. Hayashi F., et al., The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5, Nature, 410, 1099-1103, 2001.

  As general molecules capable of recognizing target molecules including pathogenic bacteria, antibodies, aptamers, peptides and the like are known. However, antibodies often require time for development, cost for production tends to be expensive, and storage stability may not be sufficient. Nucleic acid aptamers tend to have a shorter development period than antibodies, but are susceptible to heat and pH and may not have sufficient storage stability. On the other hand, peptides can be chemically synthesized and tend to have high storage stability. Therefore, an object of the present invention is to provide a microorganism-binding peptide.

The present invention includes the following aspects.
(1) At least a part of the amino acid sequence is any one of SEQ ID NOs: 1 to 25, or one or several amino acids are deleted, substituted or added in any one of the amino acid sequences of SEQ ID NOs: 1 to 25 A microorganism-binding peptide consisting of an amino acid sequence.
(2) It consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 25 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of SEQ ID NOs: 1 to 25. The microorganism-binding peptide according to (1).
(3) The microorganism-binding peptide according to (1) or (2), wherein the microorganism has a flagella.
(4) The microorganism-binding peptide according to any one of (1) to (3), wherein the microorganism is a gram-negative bacterium.
(5) The microorganism-binding peptide according to any one of (1) to (4), wherein the microorganism is Escherichia coli.
(6) A microorganism detection sensor comprising a substrate and the microorganism-binding peptide according to any one of (1) to (5) disposed on the substrate.
(7) The microorganism detection sensor according to (6), wherein the microorganism has a flagella.
(8) The microorganism detection sensor according to (6) or (7), wherein the microorganism is a gram-negative bacterium.
(9) The microorganism detection sensor according to any one of (6) to (8), wherein the microorganism is Escherichia coli.
(10) A step of bringing the microorganism detection sensor according to any one of (6) to (9) into contact with a test sample, a step of measuring the amount of microorganisms bound to the microorganism-binding peptide, A method for measuring the abundance of microorganisms in a test sample.

  According to the present invention, a microorganism-binding peptide can be provided. The microorganism-binding peptide can be used for, for example, a microorganism detection sensor.

(A) And (b) is a photograph which shows the fluorescence image of the peptide array of Experimental example 2. FIG. (A)-(c) is a photograph which shows the fluorescence image of the peptide array of Experimental example 3. FIG. (A) is a result of wild-type E. coli, (b) is a result of flagella-deficient E. coli, and (c) is a control. 10 is a graph showing a measurement result of fluorescence intensity in Experimental Example 3. (A) And (b) is a graph which shows the measurement result of the fluorescence intensity of Experimental example 5. FIG. 10 is a photomicrograph showing the results of Experimental Example 6. (A) is a photograph showing a phase contrast microscopic image of a wild-type Escherichia coli. (B) is a photograph showing a phase contrast microscopic image of Escherichia coli of a flagellar-deficient strain. (C) is a photograph showing a fluorescence microscope image of a wild-type Escherichia coli. (D) is a photograph showing a fluorescent microscope image of Escherichia coli of a flagellar-deficient strain. 10 is a photograph showing an electron microscope image of Experimental Example 7. 10 is a graph showing a measurement result of fluorescence intensity in Experimental Example 8. It is a photograph which shows the fluorescence image of the peptide array of Experimental example 10. 10 is a graph showing a measurement result of fluorescence intensity in Experimental Example 10. (A)-(d) is a photograph which shows the fluorescence image of the peptide array of Experimental example 11. FIG. (A)-(c) is a photograph which shows the fluorescence image of three independent peptide arrays, and FIG.10 (d) is a control | contrast. 14 is a graph showing a measurement result of fluorescence intensity in Experimental Example 11. (A) And (b) is a photograph which shows the result of the microscope observation of Experimental example 12. FIG. (A) is a photograph showing a phase contrast microscope image of Escherichia coli. (B) is a photograph showing a fluorescent microscope image of E. coli. It is a graph which shows the measurement result of the fluorescence intensity of Experimental example 13. It is a graph which shows the result of Experimental example 14. The result of the variant of the peptide of SEQ ID NO: 14 is shown. It is a graph which shows the result of Experimental example 14. The result of the variant of the peptide of SEQ ID NO: 15 is shown. It is a graph which shows the result of Experimental example 14. The result of the variant of the peptide of sequence number 17 is shown. It is a graph which shows the result of Experimental example 14. The result of the variant of the peptide of SEQ ID NO: 18 is shown. It is a graph which shows the result of Experimental example 14. The result of the variant of the peptide of SEQ ID NO: 19 is shown. It is a graph which shows the result of Experimental example 14. The result of the variant of the peptide of SEQ ID NO: 22 is shown. It is a graph which shows the result of Experimental example 14. The result of the variant of the peptide of SEQ ID NO: 23 is shown. It is a graph which shows the result of Experimental example 14. The result of the variant of the peptide of SEQ ID NO: 24 is shown.

[Microbe-binding peptide]
In one embodiment, the present invention provides that at least a part of the amino acid sequence is any one of the amino acid sequences of SEQ ID NOs: 1 to 25, or one or several amino acids in any of the amino acid sequences of SEQ ID NOs: 1 to 25 Provided is a microorganism-binding peptide consisting of an amino acid sequence deleted, substituted or added.

  As will be described later in Examples, the microorganism-binding peptide of the present embodiment has a high binding property to microorganisms. For this reason, it can utilize for the sensor etc. which detect a microorganism simply.

  In this specification, 1 or several may be 1-5, for example, may be 1-4, for example, may be 1-3, for example, 1-2, for example. It may be. Hereinafter, an amino acid sequence in which one or several amino acids are deleted, substituted or added in a certain amino acid sequence may be referred to as “mutant sequence”. In addition, a peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in a certain amino acid sequence may be referred to as a “peptide variant” or the like.

  The microorganism-binding peptide of the present embodiment may be a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 25 or a variant thereof. In other words, the microorganism-binding peptide of the present embodiment has a peptide consisting of any one of the amino acid sequences of SEQ ID NOS: 1 to 25 or a variant thereof as a partial peptide, and another peptide on the N-terminal side or C-terminal side. A peptide consisting of an amino acid sequence may be linked. Even such a microorganism-binding peptide has a high binding property to microorganisms.

  Moreover, the microorganism-binding peptide of this embodiment may contain one peptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 25 or a mutant sequence thereof as a partial peptide, or two or more peptides. May be. Here, the peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 25 or a mutant sequence thereof may be one type or two or more types. In addition, when the microorganism-binding peptide includes two or more types of peptides consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 25 or mutant sequences thereof, these peptides may be arranged in any order. Here, the two or more kinds of peptides may be directly connected to each other, or may be connected with another peptide interposed between the two or more kinds of peptides.

  The length of the microorganism-binding peptide of the present embodiment may be, for example, 5 to 100 amino acids, for example 5 to 50 amino acids, for example 5 to 40 amino acids, for example 5 to 30 An amino acid may be sufficient, for example, 5-20 amino acids may be sufficient, for example, 5-10 amino acids may be sufficient.

  In one embodiment, the microorganism-binding peptide of the present embodiment has one or several amino acids deleted in any one of the amino acid sequences of SEQ ID NOs: 1 to 25 or any one of SEQ ID NOs: 1 to 25, It may be a peptide consisting of a substituted or added amino acid sequence.

  In one embodiment, the microorganism to which the microorganism-binding peptide of this embodiment binds may be a microorganism having flagella. As will be described later in Examples, for example, a peptide consisting of the amino acid sequence of SEQ ID NOs: 1, 2, 4, 5, 7 to 11 or a variant thereof has particularly good binding properties to flagella.

  In the present specification, “the peptide binds to the microorganism” may be rephrased as “the peptide reacts with the microorganism”, “the peptide recognizes the microorganism”, “the peptide targets the microorganism”, and the like. it can.

  In one embodiment, the microorganism to which the microorganism-binding peptide of this embodiment binds may be a gram-negative bacterium. As will be described later in the Examples, for example, the peptide consisting of the amino acid sequence of SEQ ID NOs: 13 to 25 or a variant thereof has particularly good binding to Gram-negative bacteria.

  In one embodiment, the microorganism to which the microorganism-binding peptide of this embodiment binds may be E. coli. As will be described later in the Examples, for example, the peptide consisting of the amino acid sequence of SEQ ID NOs: 1 to 25 or a variant thereof is selected using the binding property to E. coli as an index, and thus has a good binding property to E. coli. .

[Microbe detection sensor]
In one embodiment, the present invention provides a microorganism detection sensor comprising a substrate and the above-mentioned microorganism-binding peptide disposed on the substrate.

  In the microorganism detection sensor of the present embodiment, one type of the above-mentioned microorganism-binding peptide may be arranged alone on the substrate, or two or more types may be mixed and arranged on the substrate. Further, when a plurality of microorganism-binding peptides are arranged on a substrate, a plurality of microorganism-binding peptides may be arranged on one substrate, and a plurality of substrates on which one kind of microorganism-binding peptide is arranged. It may be provided.

  Further, when a plurality of microorganism-binding peptides are arranged on one substrate, each microorganism-binding peptide may be mixed and arranged in the same region, and arranged in an array in each independent region. It may be.

  The sensor for detecting microorganisms of the present embodiment includes, for example, a surface plasmon resonance (SPR) sensor, a quartz crystal microbalance (QCM) sensor, electrochemical measurement (impedance, potential measurement, etc.) and spectroscopy. It may be a sensor based on the principle of electrochemical measurement. The microorganism detection sensor of the present embodiment can detect the presence of microorganisms in the test sample with high sensitivity.

  The SPR sensor is a sensor that detects mass change on a metal thin film with high sensitivity. First, the SPR phenomenon will be described. When light is irradiated from the light source to the back surface of the metal thin film deposited on the glass plate with a laser or the like, the light is totally reflected on the back surface of the metal thin film and at the same time an evanescent wave is generated in the metal thin film. At this time, surface plasmons are generated on the metal thin film surface side. When the wave number and frequency of the evanescent wave and the surface plasmon coincide with each other, photon energy is consumed to excite the electrons of the surface plasmon by resonance. Therefore, a phenomenon occurs in which reflected light attenuates when light is incident at a certain incident angle. This is the SPR phenomenon, and the incident angle at this time is called a resonance angle.

  The above resonance angle changes depending on the state in the vicinity of the metal thin film surface, and this change corresponds to a mass change. The SPR measurement method measures an adsorbate on the surface of a metal thin film using a change in resonance angle due to a mass change. This makes it possible to measure how many molecules the metal thin film has adsorbed on the surface.

  The QCM sensor uses the phenomenon that the resonance frequency of the crystal unit decreases according to its mass when a substance is adsorbed on the electrode surface attached to the crystal unit. It is to detect quantitatively.

  The material of the substrate may be appropriately selected according to the sensor principle or the like. For example, in the case of an SPR sensor or a QCM sensor, the substrate is made of a metal thin film. The metal species is not particularly limited, but gold (Au) is preferable because it is chemically inert and the signal generation efficiency is good.

  Examples of a method for arranging (immobilizing) the microorganism-binding peptide on the substrate include a physical adsorption method and a chemical adsorption method. In the case of the physical adsorption method, when the material of the substrate surface is a metal, it can be immobilized by the affinity between the metal and the microorganism-binding peptide. In the case of the chemical adsorption method, it is convenient to previously form self-assembled monolayers (SAMs) on the substrate surface. The self-assembled monolayer can spontaneously align and accumulate various molecules on the substrate surface, so that the microorganism-binding peptide can be immobilized on the substrate in a simple, high-density and highly-oriented manner. can do.

  For example, the self-assembled monolayer can be formed by immersing the substrate in a thiol solution. The microorganism-binding peptide may be immobilized on a carboxyl group of a thiol compound modified on the substrate surface by an amino coupling method through an amino group in the molecule, for example.

  Alternatively, a cysteine residue may be introduced in advance into the microorganism-binding peptide and immobilized on a self-assembled monolayer modified on the substrate surface by a crosslinking reaction via the thiol group of the cysteine residue. . The cysteine residue may be introduced at the N-terminus of the microorganism-binding peptide, may be introduced at the C-terminus, or may be introduced inside the peptide, but ensures a good reactivity with microorganisms. Is preferably introduced at the N-terminal or C-terminal.

  Alternatively, a chemical linker may be added to the microorganism-binding peptide and then immobilized on the substrate. Alternatively, the microorganism-binding peptide may be immobilized on the substrate using molecules that bind to each other such as biotin and avidin.

  In the microorganism detection sensor of the present embodiment, the microorganism to which the microorganism-binding peptide binds may be a flagella-containing microorganism, a Gram-negative bacterium, or E. coli.

[Method for measuring the abundance of microorganisms in a test sample]
In one embodiment, the present invention includes a step of bringing the above-described sensor for detecting a microorganism into contact with a test sample, and a step of measuring a binding amount of the microorganism to the microorganism-binding peptide. A method for measuring the abundance of microorganisms is provided. By the method of this embodiment, the abundance of microorganisms in a test sample can be measured easily and with high reliability.

  The test sample is a sample to be measured for the abundance of microorganisms, and examples thereof include beverages, foods, samples collected from the environment, and the like. Examples of the sample collected from the environment include, for example, a filter paper or the like obtained by wiping indoor walls or floors and suspended in a buffer solution.

  The step of bringing the microorganism detection sensor into contact with the test sample may be appropriately performed according to the principle of the sensor or the like. For example, when the microorganism detection sensor is an SPR sensor or a QCM sensor, the test sample may be dissolved in a buffer solution and introduced into the sensor unit. Here, it is convenient that the SPR sensor or the QCM sensor adopts a flow system. Here, the flow system refers to a system that continuously supplies liquid phase molecules to immobilized solid phase molecules under certain conditions (concentration, speed, supply amount).

  Thereby, in the microorganism detection sensor, the substrate on which the microorganism-binding peptide is arranged can be brought into contact with the test sample.

  The step of detecting whether or not a microorganism has bound to the microorganism-binding peptide may be appropriately performed according to the principle of the sensor. For example, in the case of an SPR sensor, the amount of microorganisms bound to the microorganism-binding peptide placed on the substrate may be measured by measuring the change due to the mass change of the resonance angle.

  In addition, in the case of a QCM sensor, a phenomenon is used in which when a microorganism binds to a microorganism-binding peptide arranged on the surface of an electrode attached to a crystal resonator, the resonance frequency of the crystal resonator decreases according to its mass. Then, it is good to measure the binding amount of microorganisms.

  The amount of microorganisms bound to the measured microorganism-binding peptide corresponds to the amount of microorganisms present in the test sample. Here, using a suspension of microorganisms of known concentration as a standard, the abundance of microorganisms is measured under the same conditions as the test sample, and the measured value is compared with the measured value of the test sample. It is also possible to quantify the abundance of microorganisms in the test sample.

  Next, although an experiment example is shown and this invention is demonstrated in detail, this invention is not limited to the following experiment examples.

[Experimental Example 1]
(Preparation of peptide array 1)
A peptide array consisting of a partial sequence of the amino acid sequence of the extracellular region of human TLR5 was prepared. TLR5 is known to recognize flagellin, a bacterial flagellar constituent protein. The amino acid sequence of human TLR5 is shown in SEQ ID NO: 26. The sequence consisting of the 21st to 639th amino acids of SEQ ID NO: 26 is the amino acid sequence of the extracellular region of TLR5.

  All partial sequences constituting the peptide array were peptides consisting of 8 amino acids. In the sequence consisting of the 21st to 639th amino acids of SEQ ID NO: 26, a peptide consisting of 8 amino acids from the N-terminus is defined as 1 peptide, and 2 residues are shifted from there toward the C-terminus, and the total length is 8 amino acids each. A peptide array composed of 307 types of peptides was prepared.

  First, an activated membrane was prepared. A cellulose membrane (filter paper 542, GE Healthcare) dried at 37 ° C. for 1 day was immersed in dimethylformamide (hereinafter referred to as “DMF”) for 1 day. Subsequently, the membrane was washed twice with methanol and then dried. Subsequently, DMF 300 mL, 9-fluorenylmethyloxycarbonyl (hereinafter referred to as “Fmoc”)-β-alanine 24 mL (1.0 M), diisopropylcarbodiimide (hereinafter referred to as “DIPCI”) 500 μL, 1-methyl The membrane was immersed for 2 days in an activation reagent prepared by mixing 400 μL of imidazole (hereinafter referred to as “NMI”). Finally, the membrane was washed 3 times each with DMF and methanol, and dried by applying cold air. The completed activated membrane was stored in a -20 ° C freezer.

  Subsequently, a peptide array was prepared. Using a peptide synthesizer (model “ResPep SL, KIFU20140047-0000”, Intavis), by repeating amino acid spotting at a predetermined position on the activated membrane described above, peptide elongation by Fmoc solid phase synthesis reaction Reaction was performed. As the amino acid solution to be spotted, 0.5M Fmoc amino acid solution dissolved in N-methyl-2-pyrrolidone (hereinafter referred to as “NMP”), DIPCI and hydroxybenzotriazole (hereinafter referred to as “HoBt”) are used. An activator prepared by mixing at a molar ratio of 1: 4 and mixed at a volume ratio of 17: 100 was used. For deprotection of the Fmoc group, 20% piperidine (Watanabe Chemical Co., Ltd.) was used. For capping, a solution in which acetic anhydride and DMF were mixed at a volume ratio of 2: 100 was used. Further, DMF and ethanol were used for washing the membrane.

[Experiment 2]
(Screening of microorganism-binding peptides 1)
The peptide array prepared in Experimental Example 1 was reacted with E. coli (wild strain) to screen for E. coli binding peptides.

For cultivation of E. coli, Soy Broth medium (Nippon Becton) was used. A colony formed with a solid medium was picked up and cultured in a liquid medium of about 100 mL at 37 ° C. overnight so that the OD660 exceeded 2.0, and then a binding test was performed.

  The cultured Escherichia coli was collected by centrifugation at 5000 rpm for 10 minutes, and resuspended in a phosphate buffer (hereinafter referred to as “PBS”) (pH 7.4). Turbidity was measured with a spectrophotometer (model “UV-2600”, Shimadzu Corporation), and diluted with PBS so that the bacterial solution was OD660 = 2.0. The peptide array was immersed in the suspension and incubated overnight at 37 ° C.

  Thereafter, the peptide array was washed three times with 0.05% Tween 20 (Sigma) -PBS (hereinafter referred to as “PBS-T”), and then with a fluorescent reagent SYTO9 (final concentration 1 μM, Invitrogen) at 37 ° C. for 30 minutes. Stained. Thereafter, the peptide array was washed three times with PBS-T, and a fluorescence image was obtained using an image analysis system (model “Typhoon FLA 9500BGR”, GE Healthcare). Subsequently, the measured fluorescence intensity was digitized using image analysis software Image Quant (GE Healthcare).

  1A and 1B are photographs showing fluorescence images of peptide arrays. FIG. 1 (a) shows the result of reacting E. coli with the peptide array, and FIG. 1 (b) is a control. As a result, many peptides having binding ability to E. coli were identified.

[Experiment 3]
(Screening of microorganism-binding peptides 2)
Peptides that specifically bound to flagella of E. coli were screened using a peptide array prepared in the same manner as in Experimental Example 1.

  More specifically, the binding property between the peptide array and wild-type E. coli and the binding property between the peptide array and flagella-deficient E. coli were measured in the same manner as in Experimental Example 2. As the flagellar-deficient strain, Escherichia coli lacking the fliC gene encoding the flagellar protein was used. As a control, a peptide array stained with SYTO9 without reacting E. coli was used.

  2A to 2C are photographs showing fluorescence images of the peptide array. Fig. 2 (a) shows the result of reacting wild-type E. coli with the peptide array, and Fig. 2 (b) shows the result of reacting flagella-deficient E. coli with the peptide array. Fig. 2 (c) Is a control.

FIG. 3 is a graph showing measurement results of fluorescence intensity due to flagella-specific binding. The vertical axis in FIG. 3 indicates the fluorescence intensity (relative value), and the horizontal axis indicates the peptide number. The fluorescence intensity due to flagella-specific binding was calculated by the following formula (1) using a value obtained by digitizing the fluorescence intensity (relative value) of each spot in FIGS.
Fluorescence intensity due to flagella-specific binding = (fluorescence intensity due to binding with wild strain-control fluorescence intensity)-(fluorescence intensity due to binding with flagellar-deficient strain-control fluorescence intensity) (1)

  As a result, it was revealed that the peptides in the regions indicated as region 1, region 2, and region 3 in FIG. 3 have particularly high flagella-specific binding properties. Table 1 shows the amino acid sequences of peptides with particularly high flagella-specific binding properties.

[Experimental Example 4]
(Preparation of fluorescently labeled peptide and colloidal gold labeled peptide)
Peptides consisting of the amino acid sequences of SEQ ID NOs: 1, 5 and 8 were synthesized by Fmoc solid phase synthesis and labeled with a fluorescent label or gold colloid. Fluorescein was used as the fluorescent dye.

  The colloidal gold labeling of the peptide was performed as follows. To 500 μL of 5 nm gold colloid (type “EM.GC5”, BBI Solutions), 20 μL of 10 mM peptide was added and mixed at room temperature for 2 hours using a rotator. Thereafter, the mixture was centrifuged at 8000 × g for 5 minutes, and the supernatant was removed. Subsequently, 500 μL of PBS was added and lightly sonicated after light pipetting.

[Experimental Example 5]
(Examination of reactivity of fluorescently labeled peptide with Escherichia coli 1)
The fluorescence-labeled peptide prepared in Experimental Example 4 was reacted with wild-type E. coli and flagella-deficient E. coli to examine the binding properties.

First, 500 μL of each 10 μM fluorescently labeled peptide and E. coli (1.0 × 10 ⁹ cells) were mixed at room temperature using a rotator. The mixing time was 0 minutes, 10 minutes, 30 minutes and 60 minutes. Thereafter, it was centrifuged at 6500 × g for 5 minutes. Subsequently, the supernatant was discarded, and PBS (500 μL) was added to the precipitate and suspended by pipetting. Centrifugation was performed again, the supernatant was discarded, PBS (500 μL) was added, and the mixture was suspended by pipetting. The fluorescence intensity of the suspension at a wavelength of 521 nm was measured with a fluorescence spectrophotometer.

  4A and 4B are graphs showing the measurement results of fluorescence intensity. As a result, all of the fluorescently labeled peptides consisting of the amino acid sequences of SEQ ID NOs: 1, 5 and 8 showed binding properties to E. coli. In particular, the fluorescently labeled peptide consisting of the amino acid sequence of SEQ ID NO: 8 showed higher binding to wild-type Escherichia coli than the flagella-deficient E. coli. This result shows that the peptide consisting of the amino acid sequence of SEQ ID NO: 8 has specific binding properties to flagella.

[Experimental Example 6]
(Examination of reactivity of fluorescently labeled peptide with Escherichia coli 2)
Among the samples prepared in Experimental Example 5, a sample obtained by mixing a fluorescently labeled peptide consisting of the amino acid sequence of SEQ ID NO: 8 with wild-type Escherichia coli and flagella-deficient Escherichia coli for 30 minutes is respectively a phase contrast microscope image and a fluorescence microscope Was observed.

  FIGS. 5A to 5D are photographs showing the results of microscopic observation. FIG. 5 (a) is a photograph showing a phase contrast microscopic image of a wild-type Escherichia coli. FIG. 5 (b) is a photograph showing a phase contrast microscopic image of Escherichia coli of a flagellar-deficient strain. FIG. 5 (c) is a photograph showing a fluorescence microscope image of a wild-type Escherichia coli. FIG. 5 (d) is a photograph showing a fluorescence microscope image of Escherichia coli of the flagellar-deficient strain.

  As a result, it was revealed that the fluorescently labeled peptide consisting of the amino acid sequence of SEQ ID NO: 8 has a high binding property to wild-type E. coli.

[Experimental Example 7]
(Examination of reactivity between fluorescently labeled peptide and E. coli 3)
Wild-type Escherichia coli was stained with the colloidal gold-labeled peptide having the amino acid sequence of SEQ ID NO: 8 prepared in Experimental Example 4, and observed with an electron microscope.

  First, E. coli was pre-cultured overnight using LB medium. Subsequently, main culture was performed at 37 ° C. until OD660 reached 1.0. Thereafter, 500 μL of the E. coli culture solution was centrifuged at 1000 × g for 5 minutes, the supernatant was discarded, and the precipitate was suspended by pipetting with 1000 μL of PBS to obtain an E. coli solution.

  Subsequently, 50 μL of gold colloid-labeled peptide was added to 1000 μL of E. coli solution, and mixed for 1 hour at room temperature using a rotator. Subsequently, the mixture of the E. coli solution and the colloidal gold labeled peptide was centrifuged at 6500 × g for 5 minutes. Subsequently, the supernatant was discarded, and 200 μL of PBS was added and suspended by pipetting. Thereafter, E. coli stained with colloidal gold-labeled peptide was observed with an electron microscope.

  FIG. 6 is a photograph showing an electron microscope image. As a result, gold colloid was detected in the portion surrounded by the dotted line. It was confirmed that the colloidal gold-labeled peptide consisting of the amino acid sequence of SEQ ID NO: 8 specifically binds to flagella or cilia of Escherichia coli.

[Experimental Example 8]
(Investigation of microorganism-binding peptides 1)
In the peptide consisting of the amino acid sequence of SEQ ID NO: 8, which amino acid is important for binding to E. coli was examined.

  Specifically, first, in the same manner as in Experimental Example 1, a peptide array composed of peptides in which the amino acid sequence of SEQ ID NO: 8 was substituted with alanine one amino acid at a time from the N-terminal was prepared. Subsequently, in the same manner as in Experimental Example 2, a binding assay between the prepared peptide array and E. coli (wild strain) was performed, and the fluorescence intensity was measured. FIG. 7 is a graph showing the measurement results of fluorescence intensity.

  As a result, it was shown that when the amino acid of SEQ ID NO: 8 was substituted with alanine (A), the binding property of the peptide to Escherichia coli decreased. This result indicates that these amino acids play an important role in binding to E. coli.

[Experimental Example 9]
(Preparation of peptide array 2)
A peptide array consisting of a partial sequence of the amino acid sequence of human TLR4 was prepared. TLR4 is known to recognize the endotoxin lipopolysaccharide (LPS) present in the cell wall of Gram-negative bacteria. The amino acid sequence of human TLR4 is shown in SEQ ID NO: 27.

  All partial sequences constituting the peptide array were peptides consisting of 8 amino acids. In the amino acid sequence of SEQ ID NO: 27, a peptide consisting of 8 amino acids from the N-terminus is defined as 1 peptide, and 2 residues are shifted from the N-terminal toward the C-terminus, consisting of a total of 420 peptides each having a length of 8 amino acids. A peptide array was prepared in the same manner as in Experimental Example 1.

[Experimental Example 10]
(Screening of microorganism-binding peptides 3)
The peptide array prepared in Experimental Example 9 was reacted with E. coli (wild strain), and E. coli binding peptides were screened in the same manner as in Experimental Example 2.

  FIG. 8 is a photograph showing a fluorescence image of the peptide array. FIG. 9 is a graph showing the measurement results of the fluorescence intensity of each peptide. The vertical axis in FIG. 9 indicates the fluorescence intensity (relative value), and the horizontal axis indicates the peptide number.

  In FIG. 9, the average value of fluorescence intensity of all peptides (shown as “average” in the figure), the average value of fluorescence intensity of all peptides + standard deviation (shown as “average + standard deviation” in the figure). Also, the average value of the fluorescence intensity of the peptides having the highest fluorescence intensity in the top 60 (shown as “average of the top 60 peptides” in the figure) is also shown. As a result, many peptides having binding ability to E. coli were identified.

[Experimental Example 11]
(Screening of microorganism-binding peptides 4)
In Experimental Example 10, a peptide array composed of the top 60 peptides having high reactivity with Escherichia coli was prepared in the same manner as Experimental Example 1.

  Subsequently, the prepared peptide array and E. coli (wild strain) were reacted in the same manner as in Experimental Example 2. The experiment was performed three times using three peptide arrays. As a control, a peptide array stained with SYTO9 without reacting E. coli was used.

  10A to 10D are photographs showing fluorescence images of the peptide array. FIGS. 10A to 10C show the results of reacting E. coli with three independent peptide arrays, and FIG. 10D is a control. As a result, a large number of peptides showing binding properties to E. coli were identified. Regions 1 to 3 surrounded by squares in FIGS. 10 (a) to 10 (d) indicate peptide regions that have particularly high binding to Escherichia coli.

  FIG. 11 is a graph showing the measurement results of fluorescence intensity. The vertical axis in FIG. 11 indicates the fluorescence intensity (relative value), and the horizontal axis indicates the peptide number. Regions 1 to 3 surrounded by squares in FIG. 11 indicate regions that have particularly high binding to Escherichia coli, and correspond to regions 1 to 3 surrounded by squares in FIGS. 10 (a) to 10 (d). Table 2 shows the amino acid sequences of peptides with particularly high binding to Escherichia coli.

[Experimental example 12]
(Examination of reactivity between fluorescently labeled peptide and Escherichia coli 4)
In the same manner as in Experimental Example 4, the peptide consisting of the amino acid sequence of SEQ ID NO: 24 was fluorescently labeled. Fluorescein was used as the fluorescent dye.

  The prepared fluorescently labeled peptide consisting of the amino acid sequence of SEQ ID NO: 24 was reacted with wild-type Escherichia coli in the same manner as in Experimental Example 6 and observed with a phase-contrast microscope and a fluorescent microscope to examine the binding properties.

  FIGS. 12A and 12B are photographs showing the results of microscopic observation. FIG. 12 (a) is a photograph showing a phase contrast microscope image of E. coli. FIG. 12 (b) is a photograph showing a fluorescent microscope image of E. coli.

  As a result, it was revealed that the fluorescently labeled peptide consisting of the amino acid sequence of SEQ ID NO: 24 has a high binding property to wild-type E. coli.

  In addition, from the obtained fluorescence microscopic images, many cells showing high fluorescence intensity were observed not only in the vicinity of the cell membrane of E. coli but also from specific regions within the cells. This result suggests that a molecule recognized by the peptide consisting of the amino acid sequence of SEQ ID NO: 24 is also present in the cell.

[Experimental Example 13]
(Investigation of microbial binding peptide 2)
In each peptide shown in Table 2, which amino acid is important for binding to E. coli was examined. Specifically, first, in the same manner as in Experimental Example 1, a peptide array composed of peptides in which the amino acid sequence of each peptide shown in Table 2 was substituted with alanine in order from the N-terminus was prepared.

  Subsequently, in the same manner as in Experimental Example 2, a binding assay between the prepared peptide array and E. coli (wild strain) was performed, and the fluorescence intensity was measured. FIG. 13 is a graph showing the measurement results of fluorescence intensity.

  As a result, it was shown that when a positively charged amino acid such as arginine (R), histidine (H), or lysine (K) is substituted with alanine (A), the binding ability of the peptide to Escherichia coli is particularly reduced. This result indicates that these amino acids play an important role in binding to E. coli.

[Experimental Example 14]
(Modification of microbial binding peptide)
Amino acid mutations were introduced into each peptide shown in Table 3, and changes in binding properties with E. coli were examined. Specifically, first, in the same manner as in Experimental Example 1, a peptide array composed of peptides in which amino acids shown in bold in each peptide shown in Table 3 were substituted with various amino acids was prepared.

  Subsequently, in the same manner as in Experimental Example 2, a binding assay between the prepared peptide array and E. coli (wild strain) was performed, and the fluorescence intensity was measured. 14 to 21 are graphs showing the measurement results of the fluorescence intensity. FIG. 14 shows the result of the variant of the peptide of SEQ ID NO: 14, FIG. 15 shows the result of the variant of the peptide of SEQ ID NO: 15, FIG. 16 shows the result of the variant of the peptide of SEQ ID NO: 17, Is the result of the variant of the peptide of SEQ ID NO: 18, FIG. 18 is the result of the variant of the peptide of SEQ ID NO: 19, FIG. 19 is the result of the variant of the peptide of SEQ ID NO: 22, and FIG. FIG. 21 shows the result of the variant of the peptide of No. 23, and FIG. 21 shows the result of the variant of the peptide of SEQ ID No. 24. 14 to 21, the vertical axis indicates which amino acid is substituted for the amino acid shown in bold in each peptide shown in Table 3. The horizontal axis indicates the fluorescence intensity (relative value) of each peptide.

  As a result, not only a peptide having a decreased binding ability to E. coli but also a peptide having improved binding ability to E. coli was obtained by amino acid substitution. More specifically, the peptide consisting of the amino acid sequence of SEQ ID NOs: 28 to 42, which is a variant of the peptide consisting of the amino acid sequence of SEQ ID NO: 14, has improved binding to Escherichia coli. Moreover, the peptide which consists of an amino acid sequence of sequence number 43 and 44 which is a variant of the peptide which consists of an amino acid sequence of sequence number 15 improved the binding property with respect to colon_bacillus | E._coli. Moreover, the peptide which consists of an amino acid sequence of sequence number 45-47 which is a variant of the peptide which consists of an amino acid sequence of sequence number 17 improved the binding property with respect to colon_bacillus | E._coli. Moreover, the peptide which consists of an amino acid sequence of sequence number 48-51 which is a variant of the peptide which consists of an amino acid sequence of sequence number 18 improved the binding property with respect to colon_bacillus | E._coli. Moreover, the peptide which consists of an amino acid sequence of sequence number 52-55 which is a variant of the peptide which consists of an amino acid sequence of sequence number 19 improved the binding property with respect to colon_bacillus | E._coli. In addition, the peptide consisting of the amino acid sequence of SEQ ID NO: 56, which is a variant of the peptide consisting of the amino acid sequence of SEQ ID NO: 22, has improved binding to Escherichia coli. In addition, the peptide consisting of the amino acid sequence of SEQ ID NO: 57, which is a variant of the peptide consisting of the amino acid sequence of SEQ ID NO: 23, has improved binding to Escherichia coli. Moreover, the peptide which consists of an amino acid sequence of sequence number 58-63 which is a variant of the peptide which consists of an amino acid sequence of sequence number 24 improved the binding property with respect to colon_bacillus | E._coli.

  According to the present invention, a microorganism-binding peptide can be provided.

Claims (9)

Any amino acid sequence or Ranaru of SEQ ID NO: 1 to 12 microbial binding peptides. The microorganism-binding peptide according to claim 1, wherein the microorganism is a microorganism having flagella. The microorganism-binding peptide according to claim 1 or 2 , wherein the microorganism is a gram-negative bacterium. The microorganism-binding peptide according to any one of claims 1 to 3 , wherein the microorganism is Escherichia coli. A microorganism detection sensor comprising: a substrate; and the microorganism-binding peptide according to any one of claims 1 to 4 disposed on the substrate. The sensor for detecting microorganisms according to claim 5 , wherein the microorganism is a microorganism having flagella. The sensor for detecting a microorganism according to claim 5 or 6 , wherein the microorganism is a gram-negative bacterium. The sensor for detecting microorganisms according to any one of claims 5 to 7 , wherein the microorganism is Escherichia coli. A step of bringing the microorganism detection sensor according to any one of claims 5 to 8 into contact with a test sample;
Measuring the amount of microorganisms bound to the microorganism-binding peptide;
A method for measuring the abundance of microorganisms in a test sample.