JP2012211960A - Technique for real-time visualization of substance interaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique for real-time visualization of an interaction between a substance and a substance, specifically, a plate for microscopic observations useful for real-time microscopic observation of protein interaction, a plate mounting device for microscopic observations with the plate for microscopic observations, and a method for detecting the protein interaction using the plate for microscopic observations or the plate mounting device for microscopic observations.SOLUTION: A plate 1 for microscopic observations is used for detecting an interaction between substances such as proteins. The plate is a flat plate formed of a nonluminescent resin material with optical transparency. One surface 10 of the plate is formed with at least a recess 2 constituting a fluid passage, and the base 22 of the recess is formed with a plurality of dent portions 3 capable of storing beads.

Description

  The present invention relates to a technique for visualizing the interaction of substances, particularly proteins, in real time. More specifically, a microscope observation plate useful for observing protein interactions in real time under a microscope, a microscope observation plate mounting device equipped with the microscope observation plate, and the microscope observation plate or microscope observation The present invention relates to a method for detecting a protein interaction using a plate mounting apparatus.

  In exploring the functions of interesting proteins in biological phenomena, it is no longer possible to ignore the interactions that consist of “binding” and “dissociating” with other proteins. For this reason, various methods and instruments for analyzing the interaction between proteins have been developed. For example, as a method for examining the interaction between proteins, a pull-down assay is classically known. In this method, a tagged protein (bait) immobilized on a carrier is reacted with a solution containing the protein to be measured, then the protein bound to the bait is eluted and separated, and the interaction between the two proteins is evaluated from the result. Is the method. However, according to such a method, it takes more than half a day to evaluate the protein-protein interaction, and this method has a problem that the reaction between proteins cannot be detected in real time.

  However, the devices that can detect the interaction in real time are all expensive, such as Biacore's protein interaction analysis system using the surface plasmon resonance phenomenon (SPR). There are no devices that can be visualized in real time (see, for example, Non-Patent Documents 1 and 2).

Use of surface plasmon resonance (SPR) phenomenon (GE Healthcare) <URL: http://www.biacore.com/jp/lifesciences/technology/introduction/data_interaction/index.html> Protein interaction analysis using tagged proteins (GE imagination at work) <URL: http://www.gelifesciences.co.jp/newsletter/protein_sciences/tag_interaction.asp>

  The present invention provides a method for visualizing and detecting an interaction between substances in real time at a low cost, a tool for use in the method, specifically a microscope observation plate and a microscope observation equipped with the same An object is to provide a microscope applicator such as a plate mounting device.

  The present inventors have intensively studied to solve the above problems. For example, when detecting an interaction between a substance such as a protein, one substance is bound to a bead and the other substance is fluorescent. It is possible to visually detect the binding (interaction) between a substance labeled with a dye and bound to the bead and the labeling substance by using the presence or absence of the label of the bead as an index; and further binding to the bead. A microscopic observation plate (micrometer) provided with a concave portion in which a plurality of depressions capable of accommodating (capturing) the beads are formed at the bottom using the binding (interaction) between the substance and the labeling substance as a fluid flow path. It can be detected in real time by microscopic observation by passing the reaction solution and the washing solution through the plate with a flow path) Out was.

  In particular, in the flow path (recessed portion) of the microscope observation plate placed on the microscope stage, once the substance bonded to the beads and the labeling substance are once bonded, the flow path ( Since the labeling substance once bound to the beads captured and accommodated in the depression at the bottom of the (recessed part) dissociates according to the affinity with the substance bound to the beads, the interaction between both substances (binding and dissociation) Affinity can be observed in real time.

  The present invention has been developed based on such knowledge, and includes the following embodiments.

(I) Microscopic observation plate (I-1) is a flat plate formed of a light-transmitting and non-luminous resin material,
One surface has at least one concave portion constituting a fluid flow path,
A microscope observation plate in which a plurality of recesses capable of accommodating beads are formed on the bottom surface of the recess.
(I-2) The microscope observation plate according to (I-1), wherein two or more of the concave portions are provided, and the concave portions are provided in parallel to each other.
(I-3) The microscope according to (I-1) or (I-2), wherein the beads are spherical, and the depth of the recess is smaller than ½ of the diameter of the beads Observation plate.
(I-4) The microscope observation plate according to any one of (I-1) to (I-3), wherein the resin material is processed by hydrophilic processing.
(I-5) The microscope observation plate according to (I-4), wherein the hydrophilic processing is plasma discharge treatment or corona discharge treatment.
(I-6) The microscope observation plate according to any one of (I-1) to (I-5), wherein the resin material is polydimethylsiloxane.

(II) A sample container on which the microscope observation plate according to any one of (I-1) to (I-6) can be placed inside the microscope observation plate mounting apparatus (II-1), and the sample container A support unit that can hold the storage tool and is placed on a microscope stage, and a lid that covers an upper surface side opening of the support unit,
The sample container includes a main body whose upper surface is open and a lid body that closes the upper surface of the main body, and at least two hose connection portions are integrally formed on the upper surface of the lid body or the side surface of the main body. A microscope observation plate mounting device in which a hose insertion hole into which a hose for supplying or discharging a reaction solution or a cleaning solution is inserted into the sample container is formed in the hose connection portion.

(III) A method for detecting an interaction between a substance and a substance (III-1) A method for detecting a binding between a test substance and a labeled substance,
A method of performing the following steps (a), (b) and (c) on the microscope observation plate described in any one of (I-1) to (I-6) placed on a microscope stage:
(A) a step of bringing a bead to which a test substance is bound into contact with a labeling substance in the presence of a reaction solution;
(B) a step of passing the cleaning liquid in parallel with the eyes of the concave portion of the microscope observation plate,
(C) A step of detecting a label derived from the labeling substance.
(III-2) A method for detecting the binding between a test substance and a labeling substance,
A method of performing the following steps (a ′), (b) and (c) on the microscope observation plate according to any one of (I-1) to (I-6) placed on a microscope stage:
(A ′) passing a solution containing a test substance-bound bead and a labeling substance through a microscope observation plate in parallel with the concave eye;
(B) a step of passing the cleaning liquid in parallel with the eyes of the concave portion of the microscope observation plate,
(C) A step of detecting a label derived from the labeling substance.
(III-3) The test substance or labeling substance consists of peptides, proteins, nucleic acids, saccharides, lipids, enzymes, enzyme substrates, receptors, ligands, antibodies, antigens, lectins, haptens, and other small or high molecular compounds. The method according to (III-1) or (III-2), which is at least one selected from the group.
(III-4) The method according to (III-1) or (III-2), wherein the test substance or labeling substance is a peptide or protein.
(III-5) The method according to any one of (III-1) to (III-4), wherein the test substance is a tagged substance.
(III-6) The tag of the tagged substance is at least one selected from the group consisting of a GST tag, MBP tag, His tag, Strep (II) tag, FLAG tag, HA tag, myc tag, and BCCP tag. The method described in (III-5).
(III-7) A protein having a nuclear translocation signal is used as one of the test substance or the labeling substance, and a protein that recognizes and binds to the nuclear translocation signal is used as the other substance (III- 1) The method described in any one of (III-6).
(III-8) The method according to (III-7), wherein the nuclear translocation signal is a nuclear translocation signal of NAC-1 (nucleus accumbens-associated protein 1) or a nuclear translocation signal of nucleoplasmin.
(III-9) The method according to (III-7) or (III-8), wherein the protein that recognizes and binds to a nuclear translocation signal is importin α.
(III-10) The method according to any one of (III-1) to (III-9), which is a method for evaluating an interaction between a test substance and a labeling substance.
(III-11) The method according to any one of (III-1) to (III-9), which is a method for evaluating the affinity between a test substance and a labeling substance.

  According to the method of the present invention, the interaction between substances, in particular the interaction between proteins and proteins, is observed with a microscope having a specific shape provided with a recess having a recess formed on the bottom surface as a fluid flow path. When the plate is placed on a microscope stage on a plate for use (a plate with a microchannel), it can be detected visually in real time by microscopic observation. In particular, in the flow path (recess) of the microscope observation plate placed on the microscope stage, the bead-binding substance and the labeling substance are once combined, and then passed through an arbitrary cleaning solution, whereby the flow path (recess) Since the labeling substance once bound to the beads captured and accommodated in the bottom of the bottom is dissociated according to the affinity with the substance bound to the beads, the interaction between both substances (binding and dissociation) and the affinity of both are real-time. Can be observed.

  The microscope observation plate of the present invention and the microscope observation plate mounting apparatus including the same are used to visually and in real-time the interaction between substances, particularly the interaction between proteins and proteins, using a microscope. It is useful as an applicator for a microscope that is preferably used for detection.

It is a perspective view which shows the external appearance structure of the plate for microscope observation which concerns on one Embodiment of this invention. It is sectional drawing which follows the AA line of FIG. It is sectional drawing which follows the BB line of FIG. It is a front view which shows the external appearance structure of the plate for microscope observation which concerns on other embodiment. It is sectional drawing which shows the structure of a hollow part. It is a disassembled perspective view of the plate mounting apparatus for microscope observation which concerns on one Embodiment of this invention. It is a perspective view of the sample container used for the plate mounting apparatus for microscope observation. It is a perspective view which isolate | separates and shows the sample container shown in FIG. It is sectional drawing which follows the AA line of FIG. GST fusion importin α3 (wild type) protein (labeled “wild-type” in the figure) and GST fusion importin α3 (W179A, N183A) protein (in the figure, “WN”) bound to glutathione beads by the pull-down method. → AA)) for each test sample [mCherry fusion NAC-1 (180-207) [labeled as "mC-NAC-1 (180-207)]], mCherry fusion nucleoplasmin-NLS [marked in the figure] , "MC-nucleoplasmin (NLS)"], mCherry [labeled "mC" in the figure]), then eluted and subjected to SDS-PAGE (Experimental Example 1). In the figure, the “input” lane shows the standard results obtained by subjecting each test sample (mCherry fusion NAC-1 (180-207), mCherry fusion nucleoplasmin-NLS, mCherry) itself to SDS-PAGE. In the figure, “mC fusion proteins” mean mCherry fusion NAC-1 (180-207) and mCherry fusion nucleoplasmin-NLS. It is a figure which shows the outline which performs "on beads microscope assay (the 1)" (Experimental example 2) on a cover glass. The observation data which performed "on beads microscope assay (the 1)" (experimental example 2) on a cover glass are shown. GST fusion importin α3 (wild type) protein (marked “wild-type” in the figure) and GST fusion importin α3 (W179A, N183A) protein (marked “WN → AA” in the figure) black The visible left image shows the fluorescence observation result, and the gray image on the right side shows the transmitted light observation result. In the upper, middle and lower stages, mCherry, mCherry fusion nucleoplasmin-NLS [labeled as “mCherry-nucleoplasmin (NLS)” in the figure], and mCherry fusion NAC-1 (180-207) [in the figure, “ When mCherry-NAC-1 (180-207) ”is used], the interaction with the bead-bound GST fusion importin α3 protein is shown. The result of having statistically processed the observation data of "on beads microscope assay (1)" (Experimental example 2) on a cover glass is shown. The observation data of "on beads microscope assay (the 2)" (Experimental example 3) performed by diluting on a cover glass are shown. The top two stages are a mixture of glutathione beads bound with GST fusion importin α3 (wild type) protein and mCherry fusion nucleoplasmin-NLS in the reaction mixture. PBS diluted with -X100). In the lower two rows, glutathione beads to which GST-fused importin α3 (W179A, N183A) protein is bound and mCherry-fused nucleoplasmin-NLS are mixed, and the washing solution (1% Triton-X 100 is collected while sequentially collecting the reaction solution). PBS containing PBS) and diluted. In each of the upper and lower two stages, the upper row of the figure that appears black indicates the fluorescence observation result, and the lower row of the figure that appears gray indicates the transmitted light observation result. The result of having statistically processed the observation data of "on beads microscope assay (the 2)" (Experimental example 3) performed while diluting on a cover glass is shown. It is a figure which shows the outline of the "on beads microscope assay (the 3)" (experimental example 4) on the plate for microscope observation (plate with a microchannel) of this invention. The plane portion shown in the figure means the bottom surface 22 of the flow path (recessed portion) of the microscope observation plate on which the recessed portion 3 is formed.

(I) Microscope Observation Plate FIG. 1 shows an external configuration of a microscope observation plate 1 according to an embodiment of the present invention. 2 is a cross-sectional view taken along the line AA of the microscope observation plate 1 of FIG. 1, and FIG. 3 is a view of B of the microscope observation plate 1 of FIG. 1 (hereinafter also simply referred to as “plate 1”). Cross-sectional views along line -B are respectively shown.

  The plate 1 of the present embodiment is formed in a flat plate shape with a light-transmitting and non-light-emitting resin material. Examples of such resin materials include polyethylene terephthalate, polyvinyl chloride, polymethacrylic acid, polystyrene, polycarbonate, polyethylene, polypropylene, polyvinyl acetate, nylon, polymethylpentene, polyether siphon, polysulfone, polyisobrene, polydimethylsiloxane, or These copolymers are exemplified. Polydimethylsiloxane is preferable because it is a light-transmitting and non-light-emitting resin material that can be easily processed.

  The plate 1 of this embodiment made of these resin materials is hydrophilically processed so that at least the portion of the surface that comes into contact with the specimen, specifically, the recess 2 constituting the fluid flow path in the plate 1 includes the recess 3. Preferably it has been treated. The hydrophilic processing method is not particularly limited, and examples thereof include plasma discharge treatment or corona discharge treatment.

By the way, plasma discharge treatment is an application of an electric field to gas molecules, so that free electrons existing in the gas are accelerated to have velocity energy, and the gas molecules colliding with the electrons dissociate and contain various chemical species. In this method, the surface of the material is modified by forming plasma and irradiating the surface of the material with the plasma. In the corona discharge treatment, corona discharge is generated in the atmosphere using high frequency and high voltage on the surface of the material having few polar groups, and electrons are directly irradiated onto the material surface together with the reactive groups generated thereby. This is a method for surface modification. By such processing, at least a portion of the surface of the plate 1 that comes into contact with the specimen is subjected to hydrophilic processing. Here, although the wetting tension of the surface of the plate 1 subjected to hydrophilic processing is not limited, it can be prepared to be 3.5 × 10 ^{−2 to} 9.5 × 10 ⁻² N / m. The wetting tension can be calculated by measuring the contact angle of water, or can be measured and calculated using a surface tension measuring reagent.

  On one surface 10 of this plate 1 (also referred to as “upper surface 10” for distinction from the other surface of the plate 1), there is at least one recess 2 extending in the length direction of the plate 1 (shown in FIG. 1). In the embodiment, five) are provided. The recess 2 functions as a flow path for storing and circulating a fluid such as a reaction liquid or a cleaning liquid in a state where the fluid can be circulated. It is provided so as to communicate with the part without any barrier.

  The number of the concave portions 2 formed in the plate 1 may be at least one or more, and the upper limit is not particularly limited, and may be appropriately set based on the length in the width direction of the plate 1 and the horizontal width of the concave portion 2. it can. For example, as the size of the plate 1, the length in the width direction and the length direction (width direction × length direction) is usually 10-25 mm × 10-60 mm, preferably 10-25 mm × 15-40 mm, more preferably 15-25 mm x 20-30 mm can be illustrated. The width of the recess 2 formed on one surface of the plate 1 is typically 0.8 to 2.2 mm, preferably 1 to 2 mm, more preferably 1.2 to 1.8 mm. Can do. Although not limited, the upper limit of the number of recesses 2 is 10 or less, preferably 8 or less, more preferably 7 or less, and particularly preferably 6 or less.

  In the present embodiment, on the surface 10 (upper surface 10) of the plate 1, six standing walls 20 are erected in parallel with each other at a predetermined interval, and are sandwiched between two adjacent standing walls 20. A recess 2 is formed by the upper surface 10 of the plate 1. Here, the inner bottom surface of the recess 2 formed by two adjacent standing walls 20 is not only the upper surface 10 of the plate 1 but also the bottom surface 22 of the recess 2 (see FIGS. 2 and 3). However, the present invention is not necessarily limited to this, and as shown in FIG. 4, one or more grooves 21 are formed in parallel on each other on the upper surface 10 of the flat plate 1 (in FIG. 4, three grooves are formed). 21 is formed), it is possible to form the recess 2 which becomes a flow path of a fluid such as a reaction liquid or a cleaning liquid. In this case, the bottom surface inside the recess 2 formed by forming the groove 21 is not only the bottom surface 22 of the recess 2 but also the top surface 10 of the plate 1.

  The bottom surface 22 (FIGS. 1 to 4) of each recess 2 thus prepared is formed with a plurality of recesses 3 that can accommodate beads M bound to a substance, as shown in FIG. (See FIGS. 2, 3 and 4).

  The recess 3 captures and accommodates the beads M in the recess 3 when a fluid such as a reaction solution containing the beads M is circulated in the recess 2 of the plate 1, and subsequently circulates a fluid such as a cleaning solution. In this case, the bead M is restrained in the depression 3 so that the beads M do not come out of the depression 3 due to the flow of the fluid. Therefore, the recess 3 is fitted into the recess 3 so that the bead M is captured and accommodated in the recess 3, and the bead M once captured and accommodated does not jump out of the recess 3. It is formed in a size and shape along the size of the beads M so as to be held.

  Here, the shape of the beads M is substantially spherical, preferably spherical. Moreover, although the magnitude | size is not restrict | limited, Usually, about 20-150 micrometers in diameter, Preferably it is about 30-100 micrometers, More preferably, about 50-100 micrometers can be illustrated.

  In the present embodiment, the recess 3 is formed in a truncated cone shape having a circular cross-sectional view (FIG. 5). By forming it in the shape of a truncated cone, the beads M flowing in the recess 2 together with the fluid can be easily captured and accommodated in the recess 3. In addition, the shape of the hollow part 3 is not necessarily restricted to this, The cross-sectional view shape may be formed in the shape of a truncated pyramid square. Further, in order to firmly hold the bead M once captured in the recess 3, the side peripheral surface 30 of the recess 3 is moved from the upper end toward the lower end of the recess 3 as shown in FIG. 5. Rather than inclining in the direction, the depression 3 can be formed in a columnar shape or a prismatic shape by hanging downward.

  On the other hand, the depth D of the recess 3 is preferably designed to be deeper in order to hold the bead M firmly in the recess 3. In order to attach the circular fluorescent signal S as a label, at least the upper half of the bead M needs to be exposed above the recess 3. Therefore, it is desirable that the depth D of the recess 3 is set to be smaller than ½ of the diameter r of the bead M. Preferably it is less than 1/2 and more than 1/4, more preferably less than 1/2 and 1/3 or more.

  In this embodiment, as shown in FIG. 2 and FIG. 3, each of the recesses 3 is arranged in parallel at a predetermined interval in the length direction of the recess 2 (see FIG. 3). A total of 15 depressions 3 are arranged in a matrix by providing three rows of depressions 3 in the width direction of the recesses 2 (see FIG. 2) with a predetermined interval. The fifteen hollow portions 3 are arranged so as to be densely gathered in the central portion in the length direction of each concave portion 2. The central portion in the length direction is a portion corresponding to an actual visual field region that can be observed when the plate 1 is placed on a microscope stage. That is, in the bead M captured and accommodated in the dent part 3 by arranging the dent part 3 so as to be concentrated in the central part in the length direction of the dent part 2, proteins bound to the bead M and other proteins (labels) It is possible to efficiently observe the interaction with the protein.

  However, the arrangement of the depressions 3 on the bottom surface 22 of the recess 2 of the plate 1 is not necessarily limited to this, and the number of depressions 3 provided in one row can be increased or decreased from five. It is also possible to increase or decrease the number of columns from three columns. Moreover, it is also possible to arrange | position each hollow part 3 so that the hollow part 3 may be scattered over the substantially whole area of the length direction of each recessed part 2. FIG. In this way, each recess 3 can be arranged at an arbitrary position on the bottom surface 22 of the recess 2.

  An inclined groove 4 is appropriately formed in a region where the recess 3 of the bottom surface 22 of the recess 2 is not disposed. The inclined groove 4 includes an inclined surface 40 that is inclined so as to become lower in a direction F (a direction from right to left in the example shown in FIG. 3) in which a fluid such as a reaction liquid or a cleaning liquid flows. And a vertical surface 41 formed continuously at the inclined lower end. If the beads M jump out of the recess 3 due to the flow of a fluid such as a reaction solution or a cleaning solution and move in the recess 2 by riding on the flow, the beads M are held in another recess 3. By vigorously colliding with the bead M, the bead M normally held in the recess 3 may be pushed out. This inclined groove 4 is for solving the above-mentioned problem, and by dropping the bead M that has jumped out of the recess 3 toward the bottom 42 at the lower end via the inclined surface 40, the bead M is captured. The bead M is restricted from moving in the recess 2.

  The microscope observation plate of the present invention described above is preferably used in the method described in (III) described later in a form incorporated in the following (II) microscope observation plate mounting apparatus or as it is. Can do.

(II) Microscope Observation Plate Placement Device The microscope observation plate placement device 5 includes a support unit 6, a container 7, and a lid 8, as shown in FIG. The support unit 61 has a main body 60 formed in a substantially rectangular frame shape. The main body 60 includes an outer wall 61, an inner wall 62, and a bottom wall 63. An upper surface side opening 64 is formed on the upper surface, and a lower surface side opening 65 is formed on the lower surface. A support portion 66 protruding inward is formed at the lower end portion of the inner side surface of the inner wall 62. A heater 74 is provided on the back surface of the bottom wall 62 of the main body 60. The heater 74 has a structure in which a nichrome wire is embedded in a metal plate, and is attached in close contact with the back surface of the bottom wall 62, and the lower surface side opening 65 of the bottom wall 62 is formed by the heater 74. Covered. A rectangular hole 75 is formed in the heater 74. In FIG. 6, 67 is an electric cord, and the electric cord 67 is electrically connected to the nichrome wire of the heater 74.

  The container 7 has a substantially rectangular plate-like base 70, and the base 70 is formed in such a dimension that the outer edge is placed on the support portion 66 of the main body portion 60. A hole 71 is formed at the center of the base 70, and a recess 72 is formed around the hole 71. A sample container 9 such as a dish described later is detachably mounted in the recess 72. In addition, the base 70 is provided with a pair of presser springs 73 so as to be rotatable. When the sample container 9 is provided in the recess 72, the sample container 9 is fixed by the presser spring 73.

  The lid 8 includes a substantially rectangular frame 80 and a transparent glass 81 held by the frame 80.

  In FIG. 7, the sample container 9 is made of transparent plastic or glass, and includes a main body 90 having a slightly deep circular dish shape and a lid 91 that covers the main body 90. The above-described microscope observation plate 1 is placed on the bottom surface 92 of the main body 90. The cover body 91 is integrally formed with two hose connection portions 93 protruding from the upper surface thereof. Each hose connecting portion 93 has a bowl shape and is located on the opposite side with respect to the center of the lid 91, and a hose insertion hole 94 is formed in each. The hose insertion hole 94 has one end opened on one side of the hose connection portion 93 and the other end bent in an L shape opened on the bottom surface of the lid 91. Are open toward opposite sides. One hose insertion hole 94 is provided with a supply hose 95 for supplying the reaction liquid and the cleaning liquid into the sample container 9, and the other hose insertion hole 94 is provided with the reaction liquid and the cleaning liquid in the sample container 9 outside the sample container 9. A discharge hose 96 for discharging is inserted, and the tips of the hoses 95 and 96 are drawn downward from the bottom surface of the lid 91. The other end of each hose 95, 96 extends to the inner peripheral surface of the lid 8, and the outside of the microscope observation plate mounting device 5 also extends through a hose holder 97. In the present embodiment, each hose connection portion 93 is formed on the upper surface of the lid body 91 of the sample container 9, but may be integrally formed on the side surface of the main body 90. Further, in order to supply / discharge the reaction liquid and the cleaning liquid into the sample container 9 through different routes, a hose connection section 93 for supplying / discharging the reaction liquid and a hose connection section 93 for supplying / discharging the cleaning liquid. You may make it provide at least 2 each.

(III) Method for Detecting Interaction between Substance and Substance The present invention uses the above-mentioned (I) microscope observation plate or (II) microscope observation plate mounting device provided with the plate, using a microscope. This is a method for detecting a binding between a substance and a substance in real time.

Specifically, the method includes placing the microscope observation plate or the microscope observation plate mounting device including the plate on a microscope stage, and in that state on the microscope observation plate, the following (a ) To (c). The step (c) is performed in parallel with the steps (a) and (b), or at least in parallel with the step (b).
(A) a step of bringing a bead to which a test substance is bound into contact with a labeling substance in the presence of a reaction solution;
(B) a step of passing the cleaning liquid in parallel with the eyes of the concave portion of the microscope observation plate,
(C) A step of detecting a label derived from the labeling substance.

Moreover, it may replace with the said (a) process and may perform the following (a ') process.
(A ′) A step of passing a solution containing a test substance-bound bead and a labeling substance through a microscope observation plate in parallel with the eyes of the recess.

Hereinafter, each step will be described.
(A) Process The said (a) process is a process which makes a substance and a substance contact in a reaction liquid.

  Here, the substance is a substance to be evaluated for mutual interaction. Although not limited, one substance and the other are made specific to each other by one or more actions such as electrostatic interaction, hydrogen bonding action, ionic bonding action, hydrophobic interaction or hydrophilic interaction. Substances that are expected to interact can be exemplified. Examples of specific binding include antigen-antibody binding, receptor-ligand (antagonist or agonist) binding, enzyme-substrate binding, aptamer (peptide or nucleic acid) and substance binding to it. Examples include binding, binding between a lectin and a substance having a sugar chain (saccharide, glycoprotein), binding between subunits constituting a complex, and binding between substances such as proteins in a signal transduction process.

  However, regardless of the binding mode, substances targeted by the present invention include peptides (for example, aptamers), proteins (for example, enzymes, antibodies, receptors, antigens, lectins, glycoproteins). ), Nucleic acids (for example, including polynucleotides, DNA, RNA, aptamers), saccharides (for example, including saccharides that bind to lectins), lipids, substrates for enzymes (for example, small molecules and high molecular compounds) Ligands to receptors (for example, low molecular and high molecular compounds are included), antigens and haptens for antibodies, and low molecular or high molecular compounds that can inhibit their functions by binding to proteins, etc. Is included without limitation.

  One substance is preferably a peptide or protein, and the other substance is various substances capable of binding to the peptide or protein. As described above, the various substances include, for example, substances that bind to peptide aptamers, substrates that bind to enzymes, saccharides or glycoproteins that bind to lectins, antigens and haptens that bind to antibodies, antibodies that bind to antigens, receptors Examples include a ligand that binds to the body, a partner substance that binds to a protein or the like in a signal transduction process, and the like. In addition, when one substance is a peptide or protein having a nuclear translocation signal, a peptide or protein that recognizes and binds to the nuclear translocation signal can be exemplified as the other substance.

  Here, the nuclear localization signal (NLS) is not particularly limited, but the nuclear localization signal (NLS) of NAC-1 (nucleus accumbens-associated protein 1) used in the experimental examples described later, or the nucleus of nucleoplasmin A transition signal (NLS) can be exemplified. Examples of the protein that recognizes and binds to these nuclear localization signals (NLS) include importin α, which is present in the nuclear membrane of cells and is known to carry proteins into the nucleus in an NLS-dependent manner.

  The origin of importin α is not particularly limited, and importin α derived from animals including plants and humans can be mentioned without limitation, but human-derived importin α3 is preferred. The nucleotide sequence of the human importin α3 gene is shown in SEQ ID NO: 1 in the sequence listing. In the base sequence, the region of base numbers 10-1575 is a human importin α3 (wild type) coding region (CDS region). The amino acid sequence is shown in SEQ ID NO: 2.

  As shown in the experimental examples described later, the ability to bind to NLS disappears by substituting alanine for the 179th tryptophan and the 183rd asparagine from the N terminus of the human importin α3 (wild type) ( Conti, e. Et al., Cell 94; 193-204, 1998).

  On the other hand, NAC-1 (nucleus accumbens-associated protein 1) is a nuclear transcription control protein (Nakayama, K., et al., Proc. Natl Acad. Sci. USA, 103: 18739-18744, 2006). NAC-1 has only to have a nuclear translocation signal, is not limited to human origin, and may be derived from animals other than humans. The amino acid sequence of the human NAC-1 is shown in SEQ ID NO: 5. In the amino acid sequence, the region of amino acid numbers 183 to 205 is a region corresponding to a nuclear localization signal (NLS).

  Moreover, the origin of nucleoplasmin is not particularly limited, and nucleoplasmin derived from animals including humans can be mentioned without limitation. An example is Xenopus nucleoplasmin used in the experimental examples described below. The amino acid sequence of the nucleoplasmin is shown in SEQ ID NO: 6. In the amino acid sequence, the region of amino acid numbers 155 to 172 is a region corresponding to a nuclear localization signal (NLS).

Substances targeted by the present invention may be naturally derived from peptides and proteins, or may be artificially synthesized by techniques such as chemical synthesis, fermentation, and genetic recombination. In addition, these substances may be modified with sugars, nucleic acids, and other substances (including peptides and proteins) as well as peptides and proteins, or may be fused with these substances. Furthermore, in the present invention, the peptide is used in the meaning including oligopeptide and polypeptide. Also,
In the present invention, one of the substances is preferably labeled in advance with a coloring or chemiluminescent visualization probe and prepared as a labeling substance. The visualization probe is preferably a fluorescent probe. The labeling can be performed according to a conventional method.

  By fluorescent probe is meant a compound (fluorophore) that emits light at a different wavelength when excited by exposure to light of a particular wavelength. The fluorophore is classified into a protein called a fluorescent protein and a non-protein.

  As fluorescent proteins, green fluorescent proteins such as AcGFP1 and ZsGreen1, blue fluorescent proteins such as AmCyan1, yellow fluorescent proteins such as ZsYellow1 and mBanana: DsRed-Monomer, DsRed2, DsRed-Express, DsRed-Express2, AsRed2, tdTomato, mCherry, Examples include red fluorescent proteins such as mStrawberry and mOrange; crimson fluorescent proteins such as HcRed1 without limitation. When the target substance to be labeled is a peptide or protein, the labeling substance can be prepared by fusing the fluorescent protein to the target substance (peptide or protein).

  Fluorophores classified as non-proteins are characterized by their emission profile or “color”. Green fluorophores (eg FITC and Oregon Green) are generally characterized by their emission at wavelengths in the range of 515-540 nm. Red fluorophores (eg Texas Red, Cy5, and tetramethylrhodamine) may be characterized by their emission at wavelengths generally in the range of 590-690 nm. Examples of fluorophores include, but are not limited to, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, acridine, acridine and derivatives of acridine isothiocyanate, 5- (2'-aminoethyl) Aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N- [3-vinylsulfonyl) phenyl] naphthalimide-3,5-disulfonate (Lucifer Yellow VS), N- (4-anilino-1-naphthyl) ) Maleimide, anthranilamido, brilliant yellow, coumarin, coumarin derivatives, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-trifluoromethylcoumarin (coumarin 151), cyanocine; 4 ', 6-diaminidino -2-Phenylindole (DAPI), 5 ', 5 "-Dibromopyrogallol-sulfonephthalein (bromopyrogallol red), 7-diethylamino-3- 4'-isothiocyanatophenyl) -4-methylcoumarin, 4,4'-diisothiocyanate dihydro-stilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene-2,2 ' -Derivatives of eosin such as disulfonic acid, 5- [dimethylamino] naphthalene-1-sulfonyl chloride (DNS, dansyl chloride), eosin, eosin isothiocyanate, derivatives of erythrosin such as erythrosine, erythrosine B and erythrosine isothiocyanate; Ethidium; fluorescein and 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2 ', 7'-dimethoxy-4', 5'-dichloro-6- Derivatives such as carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), QFITC (XRITC); Derivatives (fluorescence upon reaction with amines); IR144; IR1446; Malachite green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararose aniline; phenol red, B-phycoerythrin; Dialdehyde derivatives (fluorescing upon reaction with amines); derivatives such as pyrene and pyrene, pyrene butyrate, and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron.RTM. Brilliant Red 3B-A), rhodamine and 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, And sulforhodamine 101 sulfonyl Loride derivatives (Texas Red); N, N, N ′, N′-tetramethyl-6-carboxyrhodamine (TAMRA); derivatives such as tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC); riboflavin; Lanthanide chelate derivatives, quantum dots, cyanine, pyrerium dye, and squaraine are included.

  In addition, the other substance (in the present invention, referred to as “test substance” with respect to the “labeled substance”) is preferably bound in advance to the beads. Here, in the method of the present invention, the beads are used by being captured and accommodated in the recess 3 formed on the bottom surface 22 of the recess 2 in the above-described microscope observation plate of the present invention, and preferably have a spherical shape. is there. Although the size is not limited, the diameter is usually about 20 to 150 μm, preferably about 30 to 100 μm, more preferably about 50 to 100 μm.

  The material is not particularly limited. For example, silica, agarose gel, cellulose, polyethyleneimine, polystyrene, polymethacrylic acid, polymethyl methacrylate, styrene-maleic acid copolymer, magnetic substance (magnetite, and magnetite-dextran crosslinked) Examples include composites and magnetite-silica cross-linked composites), biopolymers (such as chitosan), and biodegradable polymers (such as polylactic acid).

  These beads have a surface presenting an active ester group, carboxyl group or amino group on the surface, or a ligand that recognizes and binds the tag bound to the test substance so that the test substance can be bound to the surface. Are commercially available as affinity beads or functional nano / microbeads, and can be obtained commercially.

  The test substance is preferably labeled with a tag or used in a state fused with a tag (protein tag). Here, as a tag, polyhistidine (His tag) that specifically binds to metal ions such as nickel; GST (glutathione S-transferase) (GST tag) that specifically binds to glutathione; specifically to maltose and dextrin Maltose binding protein (MBP tag) that binds; Strep (II) tag that specifically binds to StrepTactin; Biotin Carboxyl Carrier Protein (BCCP) tag (biotinylated peptide); In addition, influenza agglutinin HA (HA tag), human c -myc (myc tag), FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7 tag), human herpes simplex virus glycoprotein (HSV tag), E tag (epitope on monoclonal phage) Etc. can be exemplified. When a test substance labeled with such a tag is used as the test substance, the beads described above are labeled with a ligand that binds to the tag.

  In the step (a) of the method of the present invention, the beads to which the test substance is bound and the labeling substance described above are contacted in the reaction solution, preferably in the flow path (in the recess 2) on the microscope observation plate 1. It is a process to make. The method of contacting is not particularly limited. For example, as described in Experimental Example 4 described later, the reaction liquid containing the beads to which the test substance (test protein) is bound first is flow channel on the microscope observation plate 1. The test substance (test protein) -binding beads are passed through the inside (in the concave part 2), and the labeling substance (labeled protein) is then passed in a state where the test substance (test protein) -binding beads are captured and accommodated in the recess 3 formed on the bottom surface 22 of the concave part 2. Here, the reaction solution may be an aqueous solution having a salt concentration range, a pH range, and a temperature range in which the target substance is not denatured. Can be set. For example, when detecting and evaluating protein-protein interactions in the body, the physiological conditions (salt concentration [NaCl or KCl] of about 150 mM, pH of about 7.2 to 7.6, about 20 to 40 ° C.) or similar to it It is preferable to prepare it as an aqueous solution that meets the conditions.

  The contact time is not particularly limited and can be appropriately set within a range of 1 second to 30 minutes. In addition, such contact is performed by storing (still standing) the reaction solution containing the labeling substance in the flow path (in the recess 2) of the microscope observation plate 1 in which the beads to which the test substance is bound are captured and accommodated. The reaction solution containing the labeling substance may be continuously passed through the flow path (in the recess 2) of the microscope observation plate 1 using, for example, the microscope observation plate mounting device of the present invention. You may carry out while immersing.

(A ′) Step The step (a ′) is different from the step (a) in which a contact reaction between a substance and a substance is performed on a microscope observation plate. ) And a labeling substance are mixed in a reaction solution in a test tube or a Petri dish, and the reaction solution containing the test substance binding beads and the labeling substance is mixed in the flow path of the microscope observation plate 1 (in the recess 2). This is a process of passing through the eyes in parallel. The conditions of the reaction solution and the contact time are as described for the step (a).

  The passage of the reaction solution into the flow path (in the recess 2) of the microscope observation plate 1 may be performed manually using, for example, a pipette or the like, or the microscope observation plate mounting device of the present invention is used, for example. The reaction solution may be continuously passed through or circulated in the flow path (inside the recess 2) of the microscope observation plate 1 by use.

(B) Process The said (b) process is a process of wash | cleaning the test substance binding bead which contacted the labeling substance at the said (a) process. The labeling substance having a weak affinity for the test substance is once bound to the bead via the test substance in the step (a), but dissociates from the bead in the step (b).

  The step (b) is performed in the flow channel (in the recess 2) of the microscope observation plate 1 in which the contact and binding of the bead to which the test substance is bonded and the labeled substance are performed, in parallel with the eyes of the flow channel. The cleaning liquid can be passed through. The cleaning liquid may be passed manually using, for example, a pipette or the like, or, for example, using the microscope observation plate mounting device of the present invention, in the flow path of the microscope observation plate 1 (in the recess 2). ) May be carried out while continuously passing the washing liquid.

  Here, the washing liquid may be an aqueous solution having a salt concentration range, a pH range, and a temperature range in which the target substance is not denatured, similarly to the reaction liquid, and can be appropriately set depending on the purpose within the range. it can. For example, when detecting and evaluating the interaction between a protein and a protein in the body, it is preferable to prepare an aqueous solution under the same or similar conditions as physiological conditions.

  The time required for washing is not particularly limited and can be appropriately set within a range of 1 second to 30 minutes.

Step (c) The step (c) is a step of detecting a label derived from a labeling substance. This step is performed in parallel with the above-described steps (a) and (b) or the steps (a ′) and (b), or at least in parallel with the step (b).

  The label is preferably a fluorescent probe as described above, and the fluorescent probe of the substance labeled with the fluorescent probe can be detected by a fluorescence microscope capable of detecting fluorescence. Specific examples of the fluorescence microscope include various fluorescence microscopes such as a transmission fluorescence microscope, an epi-illumination fluorescence microscope, a confocal laser microscope, and a total reflection illumination fluorescence microscope. A confocal laser microscope is preferable.

  As described above, the microscope observation plate 1 according to the present invention has a configuration in which the depressions 3 that can capture and accommodate beads are arranged so as to be densely gathered at the center in the length direction of the depressions 2. Preferably there is. In this case, the microscopic observation plate 1 is placed on the stage of the microscope so that the central portion in the length direction of the concave portion 2 is in an actual observation region that can be observed with a microscope, and is captured and accommodated in the concave portion 3. The state in which the labeling substance is bound to the beads and the state in which the labeling substance is dissociated can be detected visually and in real time using a label (for example, a fluorescent probe) as a signal.

  As described above, the method of the present invention measures the interaction (binding, affinity) between a substance (test substance) and a substance (labeled substance) visually and in real time using a relatively simple device called a microscope. It is useful as a method that can be evaluated.

  Hereinafter, the present invention will be described in more detail using experimental examples. However, this invention is not restrict | limited at all by these Examples.

Experimental Example 1: Pull-down assay (1) Experimental method
(1-1) Preparation of GST fusion importin α3 protein
importin α is a protein that is present in the nuclear membrane of a cell and recognizes and binds to the nuclear translocation signal (NLS) present in the protein, and is known to carry the protein into the nucleus in an NLS-dependent manner. The importin α is known to lose its binding ability by mutating a highly conserved amino acid across the species present in the armadillo repeat of the C-terminal region (Goldfarb D. et al , Trends Cell Biol., 14: 505-514, 2004 and Lange A., et al., J. Biol. Chem., 282: 5101-5105, 2007). Among them, human importin α3 loses its binding ability by mutating 179th tryptophan (W) and 183rd asparagine (N) to alanine (A) from the N-terminal side (Conti, E. et al., Cell 94: 193-204, 1998).

  Hereinafter, human importin α3 wild type (importin α3 [Homo sapiens], GenBank: AAC25605.1, GI: 1928975) is referred to as “human importin α3 (wild type)” or “importin α3 (wild type)”, and The mutant is referred to as “human importin α3 (W179A, N183A)” or “importin α3 (W179A, N183A)”. The base sequence of the human importin α3 (wild type) gene is shown in SEQ ID NO: 1 in the sequence listing (NCBI Reference Sequence: NM — 002268.2). In the base sequence, the region of base numbers 10-1575 is a human importin α3 (wild type) coding region (CDS region). The amino acid sequence is shown in SEQ ID NO: 2.

(I) Preparation of GST fusion importin α3 (wild type) protein GST fusion protein expression vector pGEX-6P incorporating cDNA (amino acid sequence 65-521) of human importin α3 (GenBank: AAC25605.1, GI: 1928975) -1 (GE Healthcare Japan) (Kosugi S. et al., J. Biol. Chem., 284: 478-485, 2008) was used to transform E. coli. GST fusion importin α3 (wild type) protein was purified from E. coli lysate containing isopropylthiogalactoside (IPTG) derived GST fusion importin α3 (wild type) protein using glutathione agarose beads (Sigma).

(Ii) Preparation of GST-fused importin α3 (W179A, N183A) protein Using human importin α3 cDNA as a template, perform the overlap PCR according to the conventional method using the following primers to prepare human importin α3 (W179A, N183A) cDNA did. Transformation of E. coli using GST fusion protein expression vector pGEX-6P-1 (GE Healthcare Japan) incorporating human importin α3 (W179A, N183A) cDNA (amino acid sequence 65-521) It was. GST fusion importin α3 (W179A, N183A) protein was purified from E. coli lysate containing isopropylthiogalactoside (IPTG) derived GST fusion importin α3 (W179A, N183A) protein using glutathione agarose beads (Sigma) .

Primer
W179A N183A-S:
5′-TGT GAG CAA GCA GTG GC G GCA TTG GGA GC T ATC ATA GGT GAT GGG-3 ′ (SEQ ID NO: 3)
W179A N183A-AS:
5′-CCC ATC ACC TAT GAT A GC TCC CAA TGC C GC CAC TGC TTG CTC ACA-3 ′ (SEQ ID NO: 4).

(1-2) Preparation of test samples As test samples, "NAC-1 (180-207)" fused with red fluorescent protein mCherry, "nucleoplasmin-NLS" fused with red fluorescent protein mCherry, and as a control example Red fluorescent protein mCherry was prepared.

  Here, “NAC-1 (180-207)” is a nuclear transcriptional control protein, human NAC-1 (Nakayama, K., et al., Proc. Natl Acad. Sci. USA, 103: 18739-18744, 2006) is a peptide fragment consisting of the amino acid sequence of the 180-207 region including the nuclear localization signal (NLS) (183-205 sequence). The amino acid sequence of human NAC-1 is shown in SEQ ID NO: 5 in the sequence listing. In the amino acid sequence, the region of amino acid numbers 183 to 205 is a region corresponding to a nuclear localization signal (NLS).

  In addition, “nucleoplasmin-NLS” is a nuclear translocation signal of nucleoplasmin (Robbins, J., wt al., Cell 64: 615-623, 1991) that is already known to bind to human importin α3 (wild type). (NLS). The amino acid sequence of Xenopus nucleoplasmin is shown in SEQ ID NO: 6 in the sequence listing. In the amino acid sequence, the region of amino acid numbers 155 to 172 is a region corresponding to a nuclear translocation signal (NLS).

(1-3) Pull-down assay The GST-fused importin α3 (wild-type) protein and GST-fused importin α3 (W179A, N183A) protein prepared above were each bound to glutathione beads according to a standard method, and then each test was performed. The sample (mCherry fusion NAC-1 (180-207), mCherry fusion nucleoplasmin-NLS, mCherry) was mixed. The obtained beads were centrifuged to settle and washed several times with PBS containing 1% Triton X-100, and then an SDS sample buffer solution was added and heated to release each protein from the beads. Proteins released from the beads were separated by SDS-PAGE and identified by Coomassie staining.

(2) The result of the experiment is shown in FIG. In FIG. 11, the “input” lane shows the standard result of subjecting each test sample (mCherry fusion NAC-1 (180-207), mCherry fusion nucleoplasmin-NLS, mCherry) to the SDS-PAGE.

  As shown in lanes 5 and 6 of FIG. 11, since the bands of the test samples (mCherry fusion NAC-1 (180-207) and mCherry fusion nucleoplasmin-NLS) were detected, the nuclear translocation signal (NLS) was recognized. Both nucleoplasmin-NLS and NAC-1 (180-207) containing nuclear translocation signal (NLS) could be pulled down by the human importin α3 (wild type) binding. However, as shown in lanes 8 and 9, when importin α3 (W179A, N183A), which is a variant of human importin α3, is used instead of human importin α3 (wild type), nucleoplasmin-NLS and NAC -1 (180-207) could not be pulled down.

  Based on the above, nuclear translocation signals (NLS) such as nucleoplasmin and NAC-1 bind directly to importin α3 (wild type) protein and introduce mutations into highly conserved amino acids across species. It was confirmed that none of the importin α3 mutants (for example, importin α3 (W179A, N183A)) bind. In other words, by using an importin α3 (wild type) protein and a mutant in which amino acid sequences 179 and 183 are mutated (for example, importin α3 (W179A, N183A)), protein interaction via NLS is achieved. It was confirmed that it could be detected and evaluated.

  However, the pull-down method and the subsequent electrophoresis method are classical experimental methods for detecting and evaluating protein interactions, and the experiment requires at least half a day or more. Therefore, it is necessary to establish a method in which this can be observed in a shorter time and the protein interaction can be observed over time.

Experimental example 2: on beads microscope assay (1)
(1) Experimental method Glutathione beads (Glutathione beads to which GST-fused importin α3 (wild type) protein or GST-fused importin α3 (W179A, N183A) protein was bound, prepared in Experimental Example 1) on a commercially available cover glass) And test samples (mCherry fusion NAC-1 (180-207), mCherry fusion nucleoplasmin-NLS, mCherry prepared in Experimental Example 1) were mixed in PBS (reaction solution) containing 1% Triton X-100, It observed using the fluorescence microscope (Confocal laser microscope; Olympus company make). FIG. 11 shows an outline of the assay. It is a simple method that can be assayed in just one minute without taking a minute.

(2) Experimental results FIG. 12 shows actual observation data. The left figure shows the fluorescence observation result, and the right figure shows the transmitted light observation result. Due to the use of a confocal microscope, if there is protein interaction between the protein bound to the glutathione beads and the protein of the test sample, the fluorescence observation results from the red fluorescent protein mCherry in a circle around the beads A signal is observed. The fluorescence intensity of 12 beads is measured, and the result of statistical processing is shown in FIG. Here, as shown in lane 8 of FIG. 11, a signal indicating binding between human importin α3 (W179A, N183A) and nucleoplasmin-NLS, which was not found in the pull-down assay of Experimental Example 1, was observed ( (FIG. 12: right middle, FIG. 13: third from the right).

  From this result, it is not possible to quantitatively evaluate the interaction (binding and dissociation) between the labeled protein and the test protein simply by reacting the test sample containing the labeled protein on the beads to which the test protein is bound. There was found.

Experimental example 3: on beads microscopic assay (2)
In the experiment of Experimental Example 2, a part of a reaction solution (PBS containing 1% Triton X-100) on a commercially available cover glass for microscopic observation was collected, and a washing solution (PBS containing 1% Triton X-100) was taken as the solution. The reaction solution was repeatedly diluted by a method of adding a volume, and microscopic observation was performed.

  Actual observation data is shown in FIG. In the top two rows, mCherry fusion nucleoplasmin-NLS was mixed with glutathione beads bound with GST fusion importin α3 (wild type) protein and diluted sequentially (in the top row, the top row is the fluorescence observation result, the bottom row). Indicates transmitted light observation results). The bottom two are mCherry fusion nucleoplasmin-NLS mixed with glutathione beads bound with GST fusion importin α3 (W179A, N183A) protein, and diluted sequentially (the top row shows the fluorescence observation results, the bottom row). One row shows the observation result of transmitted light). The fluorescence intensity of 5 beads is measured and the result of statistical processing is shown in FIG.

  As can be seen from this result, the GST fusion importin α3 (W179A, N183A) protein was associated with the mCherry fusion nucleoplasmin-NLS without dilution in the same manner as the GST fusion importin α3 (wild type) protein before dilution. It was observed that the GST fusion importin α3 (wild type) protein rapidly dissociated.

  From this result, it was confirmed that the interaction (binding and dissociation) of proteins can be observed in real time under a microscope by sequentially diluting the reaction solution on the cover glass.

Experimental example 4: on beads microscope assay (part 3)
Instead of a commercially available cover glass, using the microscope observation plate (plate with a microchannel) of the present invention (see FIG. 1), glutathione beads (GST fusion importin α3 (wild type) protein prepared in Experimental Example 1), Or the interaction between GST-fused importin α3 (W179A, N183A) protein) and the test sample (mCherry-fused nucleoplasmin-NLS prepared in Experimental Example 1) using a fluorescence microscope (confocal laser microscope; manufactured by Olympus) Was observed.

  As a result of observing a series of reactions on this microscope observation plate with a fluorescence microscope in real time, when using beads bound with GST fusion importin α3 (W179A, N183A) protein as glutathione beads, mCherry fusion nucleoplasmin- NLS once bound, but quickly dissociated by passing the washing solution (the right figure of Step 3 in FIG. 16). In contrast, when GST-fused importin α3 (wild-type) protein-bound beads are used as glutathione beads, it binds to mCherry-fused nucleoplasmin-NLS, and the binding is stable despite the passage of the washing solution. It was observed that it was maintained (left figure of Step 3 in FIG. 16).

  As a result of observing a series of reactions on this microscopic observation plate in real time with a fluorescence microscope, as in Experimental Example 4, when using beads bound with GST fusion importin α3 (W179A, N183A) protein as glutathione beads Although mCherry fusion nucleoplasmin-NLS once binds, it dissociates rapidly when the washing solution is passed through, whereas GST fusion importin α3 (wild type) protein-bound beads are used as glutathione beads. In this case, it was observed that it bound to mCherry fusion nucleoplasmin-NLS and that the binding was stably maintained despite the passage of the washing solution.

  From the above, according to the microscope observation plate of the present invention, it was confirmed that the interaction between proteins can be observed and measured in real time using a simple device called a microscope.

DESCRIPTION OF SYMBOLS 1 Microscope observation plate 2 Recessed part 3 Indentation part 5 Microscope observation plate mounting apparatus 6 Support unit 7 Container 8 Lid 9 Sample container 10 Plate surface 22 Recessed bottom face 90 Main body 91 Lid body 93 Hose connection part 94 Hose insertion hole M beads

  SEQ ID NOs: 3 and 4 are primers (W179A N183A-S and W179A N183A-AS) used to create human importin α3 (W179A, N183A) cDNA using overlap PCR method using human importin α3 cDNA as a template. Means.

Claims (10)

It is a flat plate formed of a light-transmitting and non-light-emitting resin material,
One surface has at least one concave portion constituting a fluid flow path,
A microscope observation plate in which a plurality of recesses capable of accommodating beads are formed on the bottom surface of the recess.   The microscope observation plate according to claim 1, wherein two or more of the concave portions are provided, and the concave portions are provided in parallel to each other.   3. The microscope observation plate according to claim 1, wherein the beads are spherical, and the depth of the recess is smaller than ½ of the diameter of the beads.   The plate for microscope observation according to any one of claims 1 to 3, wherein the resin material is a resin material whose surface is subjected to hydrophilic processing.   The plate for microscope observation according to claim 4, wherein the hydrophilically processed resin material is polydimethylsiloxane that has been subjected to hydrophilic processing by plasma discharge processing or corona discharge processing. It is mounted in the sample container which can mount the plate for microscope observation in any one of Claims 1-5 inside, the container which can accommodate the said sample container, and the microscope stage which can hold | maintain the said container A support unit that covers the upper surface side opening of the support unit,
The sample container includes a main body whose upper surface is open and a lid body that closes the upper surface of the main body, and at least two hose connection portions are integrally formed on the upper surface of the lid body or the side surface of the main body. A microscope observation plate mounting device in which a hose insertion hole into which a hose for supplying or discharging a reaction solution or a cleaning solution is inserted into the sample container is formed in the hose connection portion. A method for detecting the binding between a test substance and a labeling substance,
A method of performing the following steps (a), (b) and (c) on the microscope observation plate according to claim 1 placed on a microscope stage:
(A) a step of bringing a bead to which a test substance is bound into contact with a labeling substance in the presence of a reaction solution;
(B) a step of passing the cleaning liquid in parallel with the eyes of the concave portion of the microscope observation plate,
(C) A step of detecting a label derived from the labeling substance. A method for detecting the binding between a test substance and a labeling substance,
A method of performing the following steps (a ′), (b), and (c) on the microscope observation plate according to claim 1 placed on a microscope stage:
(A ′) passing a solution containing a test substance-bound bead and a labeling substance through a microscope observation plate in parallel with the concave eye;
(B) a step of passing the cleaning liquid in parallel with the eyes of the concave portion of the microscope observation plate,
(C) A step of detecting a label derived from the labeling substance.   The protein according to claim 7 or 8, wherein a protein having a nuclear translocation signal is used as one of the test substance and the labeling substance, and a protein that recognizes and binds to the nuclear translocation signal is used as the other substance. Method. The nuclear translocation signal is the nuclear translocation signal of NAC-1 (nucleus accumbens-associated protein 1) or the nuclear translocation signal of nucleoplasmin, or the protein that recognizes and binds to the nuclear translocation signal is importin α.
The method according to claim 9.

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