JP6032862B2 - Cyclic GMP analysis method - Google Patents

Description

  The present invention relates to a cyclic GMP analysis method and a protein used in the method.

  Many hormones and neurotransmitters act on G protein-coupled receptors and cause various cellular activities through intracellular second messengers. In addition, the diversity of cellular activities is controlled by the temporal and spatial concentration distribution of second messengers within the cell. Therefore, it is necessary to capture the temporal and spatial changes of the second messenger in real time when analyzing intracellular information transmission related to specific cellular activities.

  Cyclic guanosine monophosphate (cyclic GMP), which is one of intracellular second messengers, acts as a signal transduction molecule for various biochemical reaction processes in vivo. So far, various physiological processes such as relaxation of cardiomyocytes, photoreceptor-transmission in the retina, electrolyte transport in the epithelium, bone growth, and neuronal activation have been controlled by cyclic GMP. It has become clear. Therefore, if the synthesis, degradation, localization, etc. of cyclic GMP in living cells are known, the mechanism of action of the circulatory system, kidney, retina, olfactory, central nervous system, etc. at the cellular and tissue levels can be clarified It is expected that it will be possible to obtain knowledge about drugs that regulate the intracellular cyclic GMP concentration.

  Conventionally, the measurement of cyclic GMP is performed on a large number of cells using a radiolabeled compound. In recent years, several cyclic GMP biosensors have been found, and cyclic GMP can be measured at the cellular level.

  For example, in Patent Document 1 and Non-Patent Document 1, cyclic GMP-dependent protein kinase (PKG) is utilized using fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP). A cyclic GMP analysis method for imaging using a gene in which an Iα gene and a fluorescent protein gene are fused is disclosed. Specifically, YFP and CFP sequences are fused to both ends of the PKGIα gene sequence and expressed in cells. Under normal conditions, no fluorescence of YFP is observed because FRET does not occur even when CFP is excited. On the other hand, when the cyclic GMP in the cell increases, the three-dimensional structure of PKGIα changes and FRET occurs, so that the fluorescence of the acceptor YFP is observed. It has been clarified that the change in the concentration of cyclic GMP can be observed by observing the FRET signal.

  Patent Document 2 discloses a method for quantitative analysis of cyclic GMP and cyclic AMP concentrations, which are intracellular cyclic nucleotides, using a certain CNG channel and calcium-sensitive photoprotein. In this method, specific ion channels (CNG channels) activated by cyclic nucleotides and calcium-sensitive photoproteins are expressed in cells. At this time, when the cyclic GMP or the cyclic AMP in the cell increases, the CNG channel is opened, and thereby calcium ions flow in. It has been shown that the inflowed calcium can be detected by a calcium sensitive luminescent indicator such as Fura-2 or Flou-3, or a calcium sensitive photoprotein such as aequorin or oberin.

  Non-Patent Document 2 discloses a cyclic GMP-specific phosphodiesterase 5A (PDE5A) cyclic GMP binding domain gene that utilizes luminescence resonance energy transfer (BRET) between a blue photoprotein and a green fluorescent protein (EGFP). A cyclic GMP analysis method in which a photoprotein gene and a fluorescent protein gene are fused and observed is disclosed. Blue luminescence is observed when coelenterazine as a luciferase substrate is added to cells expressing a fusion of PDE5A cyclic GMP binding domain gene, blue luminescent protein Renilla luciferase gene and EGFP gene . When intracellular cyclic GMP increases in this cell, the three-dimensional structure of the cyclic GMP binding domain changes, causing a luminescence-fluorescence resonance energy shift (BRET) from luciferase luminescence to EGFP fluorescence, and EGFP fluorescence is detected. The It has been clarified that a change in the concentration of cyclic GMP can be detected by detecting this BRET signal.

  Patent Document 3 discloses a protein in which a cyclic GMP binding domain of human cyclic GMP-activated protein kinase-derived B domain (GKI-B) or human phosphodiesterase 2A (PDE2A) and firefly luciferase substituted with circular permutation are used. A method for quantitatively analyzing the intracellular cyclic GMP concentration by using it is disclosed. In this method, luciferin serving as a luciferase substrate is added to a cell expressing a protein in which a cyclic GMP binding domain gene of GKI-B or PDE2A and a circularly permuted firefly luciferase gene are expressed. Is measured. When the cyclic GMP in the cell increases, the three-dimensional structure of the cyclic GMP binding domain changes, and the luminescence of luciferase disappears or is enhanced. It has been clarified that a change in the concentration of cyclic GMP can be detected by detecting this luminescent signal.

Japanese Patent No. 3644320 Special table 2006-520203 Special table 2009-532063

Honda A, Adams SR, Sawyer CL, Lev-Ram V, Tsien RY, Postmann WRG. “Spatiotemporal dynamics of guanosine 3 ', 5'-cyclic mono phosphatically fed es the ce s e s e n e s e n e s e n e n e n e n e,” 98, no. 5, pp. 2437-2442, 2001 Biswas KH, Society S, Visweriah SS. “The GAF domain of the cGMP-binding, cGMP-specific phosphoesterase (PDE5) is a sensor and a sink for cGMP.” Biochemistry, Vol. 47, pp. 3534-3543, 2008

  In the method using the PKG enzyme as disclosed in Patent Document 1 or Non-Patent Document 1, since the full length or enzyme domain of PKG is included in the cyclic GMP binding site of the reporter, the reporter has phosphorylase activity. There was a problem of affecting the information transmission in the cell. In addition, since fluorescent proteins are used, there is a problem that unspecific proteins existing in the cells are excited by the influence thereof to generate fluorescence (autofluorescence), and the signal / noise ratio is lowered. Furthermore, there is a problem that the range of available measurement objects is narrow due to the narrow dynamic range.

  In the method using a CNG channel and a calcium-sensitive photoprotein as disclosed in Patent Document 2, it is necessary to introduce two or more types of genes simultaneously, and there is a problem that introduction into cells and expression efficiency are low. It was. In addition, in this method, since calcium ions flow into the cell, there is a problem in that the information state in the cell is affected and the original state of the cell may not be measured.

  In the method using BRET of a photoprotein and a fluorescent protein as disclosed in Non-Patent Document 2, measurement is performed only with a luminometer or photon counting, so measurement in a single cell is not optimized or There is a problem that it is impossible and the range of available measurement methods is narrow. In particular, since the BRET conversion efficiency is low and the signal is small, a signal / noise ratio that can be used for imaging cannot be obtained.

  In the method using a fusion protein of a circular permutation luciferase and a cyclic GMP binding domain of GKI-B or PDE2A as disclosed in Patent Document 3, measurement is performed only by a luminometer or photon counting. Measurement in cells is not optimized or impossible, and there is a problem that the range of available measurement methods is narrow. In particular, since the amount of luminescence from the fusion protein is small, a signal / noise ratio that can be used for imaging cannot be obtained.

  The present invention has been made in view of the above-described problems of the prior art, and an object thereof is to provide a cyclic GMP analysis method capable of accurately quantifying the original state of a cell. In particular, accurate quantitative results from each cell can be obtained without affecting intracellular information transmission. As a result, changes in cyclic GMP concentration in individual cells can be observed over time with good reproducibility. An object of the present invention is to provide a cyclic GMP analysis method that can be used.

  According to one embodiment of the present invention, a cyclic GMP sensor protein comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, and a cyclic GMP binding region, wherein the cyclic GMP binding region is A cell production step for producing a cell containing a cyclic GMP sensor protein disposed between the N-terminal side fragment and the C-terminal side fragment, and a predetermined amount from the outside of the cell to the cell produced in the cell production step. An addition step of adding a luminescent substrate; an imaging step of taking a luminescent image of the cell to which the predetermined luminescent substrate is given in the addition step; and the intracellular light based on the luminescent image taken in the imaging step. And an analysis step of analyzing the concentration of the cyclic GMP in the cell.

  According to another embodiment of the present invention, there is provided a cyclic GMP sensor protein comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, and a cyclic GMP binding region, wherein the cyclic GMP A cyclic GMP sensor protein is provided in which the binding region is disposed between the N-terminal fragment and the C-terminal fragment.

  According to the present invention, information related to intracellular cyclic GMP can be obtained accurately and quantitatively from individual cells, and as a result, the intracellular cyclic GMP concentration can be observed with good reproducibility in individual cells. Can do.

1 is a diagram illustrating an example of an overall configuration of a light emission observation system 100. FIG. 2 is a diagram illustrating an example of a configuration of a light emission image capturing unit 106 of the light emission observation system 100. 2 is a diagram illustrating an example of a configuration of a light emission image capturing unit 106 of the light emission observation system 100. FIG. 2 is a block diagram illustrating an example of a configuration of an image analysis device 110 of the light emission observation system 100. FIG. It is a figure which shows the detection principle of cGMP using a split luciferase assay method. It is a graph which shows the light-emission quantity measured with the luminometer about various cyclic GMP sensor proteins. It is a figure which shows the luminescence image before 8-Br-cGMP stimulation in HEK293 cell which introduce | transduced GL4_N_PDE5A_GAFa1_C. It is a figure which shows the luminescence image 5 minutes after 8-Br-cGMP stimulation in HEK293 cell which introduce | transduced GL4_N_PDE5A_GAFa1_C. It is the figure which showed the time-dependent change of the emitted light intensity of GL4_N_PDE5A_GAFa1_C by 8-Br-cGMP stimulation in the selected cell. It is a figure which shows the luminescence image before 8-Br-cGMP stimulation in HEK293 cell which introduce | transduced GL4_N_PDE5A_GAFa7_C. It is a figure which shows the luminescence image 5 minutes after 8-Br-cGMP stimulation in HEK293 cell which introduce | transduced GL4_N_PDE5A_GAFa7_C. It is the figure which showed the time-dependent change of the emitted light intensity of GL4_N_PDE5A_GAFa7_C by 8-Br-cGMP stimulation in the selected cell. It is a figure which shows the luminescence image before 8-Br-cGMP stimulation in HEK293 cell which introduce | transduced cpGL4_C_PDE5A_GAFa7_N. It is a figure which shows the luminescence image 5 minutes after 8-Br-cGMP stimulation in HEK293 cell which introduce | transduced cpGL4_C_PDE5A_GAFa7_N. It is the figure which showed the time-dependent change of the emitted light intensity of cpGL4_C_PDE5A_GAFa7_N by 8-Br-cGMP stimulation in the selected cell. It is a figure which shows preparation of various cyclic GMP sensor proteins.

[Method]
The method according to the present invention will be described below.
The present invention relates to a cyclic GMP sensor protein comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, and a cyclic GMP binding region, wherein the cyclic GMP binding region and the N-terminal side fragment A cell production step for producing a cell containing a cyclic GMP sensor protein arranged between the C-terminal fragment, and an addition for adding a predetermined luminescent substrate from the outside of the cell to the cell produced in the cell production step An imaging step of capturing a luminescence image of the cell to which the predetermined luminescent substrate is given in the addition step, and the cyclic GMP in the cell based on the luminescence image captured in the imaging step. The present invention relates to an intracellular cyclic GMP analysis method including an analysis step of analyzing a concentration.

  Briefly, the present invention introduces a cyclic GMP sensor protein into a target cell, induces luminescence by giving a luminescent substrate, images the state, and specifies cGMP concentration based on the captured image. Regarding the method.

  According to the present invention, the state of cyclic GMP in a cell can be analyzed. In particular, it is possible to quantitatively measure the intracellular cyclic GMP concentration. In addition, since measurement can be performed by imaging, measurement can be performed for each cell, and further, measurement can be performed by focusing on the structure of an organelle in a cell. Furthermore, by measuring the same measurement region at regular intervals, changes in cyclic GMP concentration with respect to stimulation and temporal changes can be analyzed. Differences between cells can be analyzed by measuring each cell.

  In the cell production process, cells containing a cyclic GMP sensor protein are produced. Cyclic GMP sensor protein is a protein in which two polypeptide fragments of a luminescent enzyme, a polypeptide fragment capable of binding to cyclic GMP, and optionally other polypeptide fragments are fused, and the presence of cyclic GMP Depending on the protein, the activity of the luminescent enzyme disappears or recovers. Details of the cyclic GMP sensor protein will be described later. There are no limitations on the types of cells to be used, and bacterial cells, yeast cells, plant cells, animal cells, and the like can be used. When animal cells are used, particularly mammalian cells are used, for example mouse cells, monkey cells and human cells. The cell may be a cell aggregate cultured as a part of the living tissue, or may be in the form of an organ or the like to which the living tissue belongs or a living individual. There is no particular limitation on the method for introducing the cyclic GMP sensor protein into the cell, and a known introduction method can be used. One method is a method in which a nucleic acid containing a base sequence encoding the protein is introduced into a cell, and then the protein is expressed in the cell. For example, an expression vector containing the nucleic acid can be introduced into cells by the calcium phosphate method, lipofection method, electroporation method, or the like, and the protein can be expressed from the expression vector. Instead of expressing from a vector, the protein can also be expressed from the genome after preparing a cell in which the nucleic acid is integrated into the genome. Another method is to introduce the protein purified outside the cell directly into the cell. For example, the protein can be directly injected into cells by a microinjection method. Alternatively, cells can be incubated in a culture solution containing the protein, and the protein can be taken into the cell by endocytosis.

  In the addition step, a luminescent substrate is added to the cells produced in the cell production step. The method according to the present invention uses a luminescent reaction by a luminescent enzyme, but a luminescent substrate is required to cause this luminescent reaction. The luminescent substrate is selected according to the luminescent enzyme used. Details of the luminescent substrate will be described later. There is no special limitation in the method of adding a luminescent substrate, and it can select suitably. For example, by adding to a culture solution, a luminescent enzyme can be taken into cells via the culture solution. Alternatively, the luminescent substrate can be directly injected into the cell by the microinjection method. The timing of addition of the luminescent substrate can also be selected as appropriate. For example, a luminescent substrate can be added before, simultaneously with, or after introduction of the cyclic GMP sensor protein into the cell. For example, a luminescent substrate can be included in a culture solution for handling cells through a series of operations. Alternatively, the protein and the luminescent substrate can be simultaneously injected into cells by microinjection. Preferably, the luminescent substrate is added immediately before the measurement. There is no particular limitation on the amount of the luminescent substrate to be added, but preferably the luminescent enzyme is added in an amount sufficient to cause a luminescent reaction.

  In the imaging step, an image of a cell in which a luminescent reaction has occurred is acquired. Imaging can be performed at an arbitrary timing, but is preferably performed after a certain time from the addition of the substrate. In particular, when a plurality of measurements are repeated, it is preferable to capture images at the same timing through a plurality of measurements in order to suppress variations in measurement results. The best imaging time is appropriately selected according to the conditions. When the emission intensity is low, the imaging time (exposure time when using a microscope) is lengthened, and conversely, when the emission intensity is too high, the imaging time is shortened. For example, the exposure time can be 5 to 10 seconds. Imaging can be performed by acquiring only light having a wavelength related to the luminescence reaction, but at the same time, a bright field image or an image using light having another wavelength may be acquired. For example, a specific protein in which a probe (for example, a fluorescent or luminescent protein) is fused simultaneously with a cyclic GMP sensor protein is introduced into a cell, and light from the specific protein is detected simultaneously with light derived from the cyclic GMP sensor protein. May be. An apparatus used for imaging will be described later.

  In the analysis step, analysis of the intracellular cyclic GMP concentration and the like is performed based on the acquired image. In the analysis, the amount of light emission may be converted into a numerical value by using software that functions on a computer. By using the numerical value, calculation of the ratio with the standard, comparison between samples, and the like are appropriately performed. By such processing, the intracellular cyclic GMP concentration is specified. In addition, the specification of concentration may be a rough specification such as whether or not cyclic GMP is present in a sample or whether or not it is within a set concentration range, without specifying a detailed numerical value. Good. An apparatus used for analysis will be described later.

  The present invention also relates to the above-described intracellular cyclic GMP analysis method, wherein the imaging step includes repeatedly capturing a luminescent image. In this method, imaging is repeated at regular intervals, or imaging is performed at a specific time. By taking images over time in this way, observation by time lapse or observation by moving images becomes possible. A luminescent substrate may be added for each imaging. By this method, the spatiotemporal measurement in the same cell can be performed. That is, it is possible to measure a change in cyclic GMP concentration according to time or according to stimulation. Further, by acquiring a bright field image at the same time, the relationship between the cyclic GMP concentration change and the cell shape change can be known. Moreover, the relationship between the protein and cyclic GMP can be known by expressing the protein with the probe and observing the state of the probe simultaneously.

  The present invention also relates to an intracellular cyclic GMP analysis method in which an imaging step is simultaneously performed on a plurality of different cells and an analysis step is performed for each cell. In this method, images of a plurality of different cells are acquired in one imaging. Further analysis is then performed on each cell. Analysis for each cell can be performed for a plurality of cells included in one captured image, or analysis for each cell can be performed for a plurality of cells included in each of the plurality of captured images. . Further, the imaging can be performed over time as described above. By such a method, a cyclic GMP concentration change in an individual cell can be analyzed in spatio-temporal. For example, it can be used for comparison of cyclic GMP concentrations between cells placed under the same conditions.

[Cyclic GMP sensor protein]
Next, the cyclic GMP sensor protein according to the present invention will be described. The present invention relates to the above method utilizing a cyclic GMP sensor protein, and also relates to the cyclic GMP sensor protein itself.

  The cyclic GMP sensor protein according to the present invention includes an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of a luminescent enzyme, and a cyclic GMP binding region, and the cyclic GMP binding region includes an N-terminal fragment and a C-terminal. It is arranged between the side pieces. The protein loses or recovers the activity of the luminescent enzyme depending on the presence of cyclic GMP.

  In the cyclic GMP sensor protein according to the present invention, the cyclic GMP binding region can be derived from a cyclic GMP-specific phosphodiesterase. Thereby, the cyclic GMP sensor protein can bind in a cyclic GMP-dependent manner.

  Cyclic GMP-specific phosphodiesterase is an enzyme that promotes a reaction that hydrolyzes the phosphodiester bond of cyclic GMP to produce GMP. An example of this is phosphodiesterase 5A (PDE5A). The cyclic GMP-specific phosphodiesterase has two sites that can bind to cyclic GMP, and these sites can be used as a cyclic GMP binding region. The biological species from which the cyclic GMP-specific phosphodiesterase is derived is not particularly limited as long as the phosphodiesterase has a cyclic GMP binding region, but the phosphodiesterase derived from mammals, particularly those derived from humans or mice. Phosphodiesterases are preferred.

  The cyclic GMP binding region may be a specific region in mouse cyclic GMP-specific phosphodiesterase 5A. The start position in the amino acid sequence of the “specific region” may be the 121st, 131st or 155th amino acid, and the end position is 308th, 320th, 330th, 343rd, 358th, 366th Or it may be the 384th amino acid.

  In particular, in the cyclic GMP sensor protein according to the present invention, the cyclic GMP binding region has amino acid numbers 121 to 330, 131 to 320, 131 to 330, 131 of mouse cyclic GMP-specific phosphodiesterase 5A. It may contain the amino acid sequence from position 343, or position 131 to 358. As a result, an effect of binding in a cyclic GMP-dependent manner while maintaining a high luciferase activity in mammalian cells can be obtained.

  In addition, regarding the length of the cyclic GMP binding region, the number of amino acid residues may be 150 to 260. Furthermore, the number of amino acid residues may be 190 to 230. In particular, the number of amino acid residues is preferably 190, 200, 210, 213 or 228.

  As the cyclic GMP binding region, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 20, 24, 26, 27 or 28 can be preferably used.

  SEQ ID NO: 19 shows the amino acid sequence of cyclic GMP-specific phosphodiesterase 5A (PDE5A) derived from mouse. In addition, SEQ ID NOs: 20 to 30 show the amino acid sequences of the polypeptide fragments of the mouse-derived PDE5A, which are respectively PDE5A_GAFa1, PDE5A_GAFa2, PDE5A_GAFa3, PDE5A_GAFa4, PDE5A_GAFa5, PDE5A_GAF5, PDE5A_GAF5, PDE5A_GAFA6 It corresponds to the amino acid sequence of a polypeptide fragment called PDE5A_GAFa11.

  In the present application, the luminescent enzyme means an enzyme that catalyzes a reaction involving luminescence. Luciferase can be used as the luminescent enzyme. As the luciferase, a firefly-derived one, a Renilla-derived one, a bacteria-derived one or the like can be used, and it is particularly preferable to use a firefly-derived luciferase. The luminescent substrate is selected according to the luminescent enzyme. When luciferase is used as the luminescent enzyme, luciferin can be used as the luminescent substrate.

  The luminescent enzyme contained in the cyclic GMP sensor protein is contained in the protein as an N-terminal fragment and a C-terminal fragment. The “N-terminal fragment” means a fragment of a luminescent enzyme from which a plurality of amino acids have been deleted from the C-terminus, and has lost the luminescent enzyme activity. The “C-terminal fragment” means a fragment of a luminescent enzyme from which a plurality of amino acids have been deleted from the N-terminus, and has lost the luminescent enzyme activity. A cyclic GMP binding region is arranged between the N-terminal fragment and the C-terminal fragment, and if the conditions are satisfied, the N-terminal fragment and the C-terminal fragment of the luminescent enzyme in the cyclic GMP center protein There is no limitation on the arrangement. That is, the cyclic GMP sensor protein can be used in which the N-terminal fragment of the luminescent enzyme is arranged on the N-terminal side and the C-terminal fragment of the luminescent enzyme is arranged on the C-terminal side of the cyclic GMP sensor protein (see FIG. 5a). Alternatively, it is possible to use a product in which the C-terminal fragment of the luminescent enzyme is arranged on the N-terminal side of the cyclic GMP sensor protein and the N-terminal fragment of the luminescent enzyme is arranged on the C-terminal side of the cyclic GMP sensor protein (see FIG. 5b). The latter cyclic GMP sensor protein is given a term such as “mutant” or “cp (circular permutation)”, and is appropriately distinguished from the former.

  In the cyclic GMP sensor protein, each luminescent enzyme fragment does not have a full-length sequence, so that its activity is lost in a normal environment (for example, in an environment where no cyclic GMP is present). The activity is restored under a specific environment (for example, in an environment where cyclic GMP exists). For example, cyclic GMP binds to the cyclic GMP binding region, and the structure of the cyclic GMP binding region changes. As a result, the separated N-terminal fragment and C-terminal fragment bind to each other. Activity is restored. There is no limitation on the mode of this “binding”, and chemical bonding such as covalent bonding or non-covalent bonding may be used, but to the extent that the N-terminal fragment and the C-terminal fragment can perform the original function of luciferase. It is preferable that the state is “approaching” or “contacting”. Alternatively, the N-terminal fragment and the C-terminal fragment may be “associated”. That is, the term “binding” used in the expression includes the meaning of “approach”, “contact”, or “meeting”.

  There is no limitation on the position where the luminescent enzyme is divided, but when luciferase is used as the luminescent enzyme, the N-terminal fragment contains the first to 400th amino acids of luciferase, and the C-terminal fragment Preferably contains the 401st to 550th amino acids of luciferase. Further, the N-terminal fragment contains the 1st to 416th amino acids of luciferase, and the C-terminal fragment contains the 399th to 550th amino acids of luciferase. , Those partial sequences may overlap.

  In particular, in the cyclic GMP sensor protein according to the present invention, the luminescent enzyme is firefly luciferase, the N-terminal fragment contains the amino acid sequence of amino acid numbers 1 to 416 of firefly luciferase, and the C-terminal fragment is firefly luciferase. It preferably contains the amino acid sequence from amino acid number 399th to 550th. As a result, light is emitted only when the N-terminal fragment and the C-terminal fragment of the luminescent enzyme reassociate, so that the effect of a large signal / noise ratio is obtained.

  Furthermore, the cyclic GMP sensor protein according to the present invention may be a cyclic GMP sensor protein comprising the amino acid sequence shown in SEQ ID NO: 31, 35, 37, 48, 38 or 39. These sequences are the cyclic GMP sensor proteins GL4_N_PDE5A_GAFa1_C, GL4_N_PDE5A_GAFa5_C, GL4_N_PDE5A_GAFa7_C, cpGL4_C_PDE5A_GAFa7_N, and GL4_N4A9

  As described above, the cyclic GMP sensor protein according to the present invention loses or recovers the activity of the luminescent enzyme depending on the presence of cyclic GMP. In addition, the method according to the present invention utilizes such characteristics of the protein. In the conventional technology using intermolecular energy resonance (FRET, BRET, etc.), the amount of luminescence is small due to the low efficiency of energy resonance, and the presence / absence of cyclic GMP due to the difference in detected wavelengths. Absence is analyzed. In contrast, in the present invention, since a simple reaction by a luminescent enzyme such as luciferase is used, the amount of luminescence is large and imaging is possible, and the presence / absence of cyclic GMP corresponds to the occurrence / non-occurrence of luminescence. Therefore, a detection result with a high signal / noise ratio can be obtained.

[Nucleic acid]
Next, a nucleic acid containing a gene encoding the cyclic GMP sensor protein according to the present invention will be described.

  The nucleic acid includes a gene encoding the cyclic GMP sensor protein according to the present invention. In particular, it includes the ORF region of such genes. The codon for this gene may be optimized for expression in the cell of interest. In addition, a sequence for increasing the expression level, such as a Kozak sequence, may be included or added.

  The present invention also relates to a vector containing the nucleic acid. The vector may be an expression vector for expression in a target cell. In the case of an expression vector, in addition to the sequence of a gene encoding a cyclic GMP sensor protein, a sequence included in a general expression vector such as a sequence for controlling expression and a sequence of a marker gene may be included. The expression vector can be used in the cell preparation step of the intracellular cyclic GMP analysis method according to the present invention. That is, the expression vector can be introduced into a cell to express a cyclic GMP sensor protein and used for detection of cyclic GMP.

[apparatus]
Next, a light emission observation system 100 used in the cyclic GMP analysis method (particularly the imaging process and the analysis process) according to the present invention will be described with reference to FIGS. FIG. 1 is a diagram illustrating an example of the overall configuration of the light emission observation system 100. FIG. 2 is a diagram illustrating an example of the configuration of the light emission image capturing unit 106 of the light emission observation system 100. FIG. 3 is a diagram illustrating an example of the configuration of the light emission image capturing unit 106 of the light emission observation system 100.

  First, as shown in FIG. 1, the luminescence observation system 100 arranges a container 103 (specifically, a petri dish, a slide glass, a microplate, a gel support, a fine particle carrier, etc.) containing the cells 102 and the container 103. The stage 104, the light emission image capturing unit 106, and the image analysis device 110 are configured. A light emission image capturing unit 106 for measuring weak light emission may be disposed below the stage 104. Thereby, disturbance light from above the sample due to opening and closing of the cover can be completely blocked, and the S / N ratio of the luminescent image can be increased. The light emission image capturing unit 106 may be a laser scanning optical system.

  Here, the cell 102 is, for example, a living cell that is luminescence-labeled so as to emit light depending on the cyclic GMP concentration. In addition to a fusion protein in which a cyclic GMP-binding protein is inserted between an N-terminal luminescent enzyme and a C-terminal luminescent enzyme as a protein that emits light depending on the cyclic GMP concentration, for example, JP 2009- You may use the fusion protein currently disclosed by 532063 gazette. Also, here, the expression of the fusion protein is expressed in the cell 102 by introducing into the cell 102 an expression vector containing the polynucleotide encoding the fusion protein, which is configured to express the fusion protein. May be. In the addition step, the cell 102 is given a predetermined luminescent substrate (for example, luciferin) and a predetermined stimulus (for example, drug stimulus) from the outside of the cell.

Further, the luminescent image capturing unit 106 is specifically an upright luminescent microscope, and captures a luminescent image of the cell 102. As shown in the figure, the luminescent image capturing unit 106 includes an objective lens 106a, a dichroic mirror 106b, a CCD camera 106c, and an imaging lens 106f. Specifically, the objective lens 106a has a value of (numerical aperture / magnification) ² of 0.01 or more. The dichroic mirror 106b is used when the light emitted from the cell 102 is separated by color and the light emission amount and light emission intensity are measured by color using two colors of light emission. The CCD camera 106c takes a light emission image and a bright field image of the cell 102 projected onto the chip surface of the CCD camera 106c via the objective lens 106a, the dichroic mirror 106b, and the imaging lens 106f. The CCD camera 106c is connected to the image analysis device 110 so as to be able to communicate with each other by wire or wirelessly. Here, when there are a plurality of cells 102 in the imaging range, the CCD camera 106c may capture a light-emitting image and a bright-field image of the plurality of cells 102 included in the imaging range. The imaging lens 106f forms an image (specifically, an image including the cell 102) incident on the imaging lens 106f via the objective lens 106a and the dichroic mirror 106b. Note that FIG. 1 shows an example in which a light emission image corresponding to two light emissions separated by the dichroic mirror 106b is separately captured by the two CCD cameras 106c. The image capturing unit 106 may include an objective lens 106a, a single CCD camera 106c, and an imaging lens 106f.

  Here, when measuring the light emission amount and the light emission intensity for each color using the light emission of two colors, as shown in FIG. 2, the light emission image pickup unit 106 includes an objective lens 106a, a CCD camera 106c, and a split image unit 106d. And the imaging lens 106f. The CCD camera 106c may capture a light emission image (split image) and a bright field image of the cell 102 projected onto the chip surface of the CCD camera 106c via the split image unit 106d and the imaging lens 106f. The split image unit 106d separates the light emitted from the cell 102 by color, and is used when measuring the light emission amount and light emission intensity by color using two colors of light, similar to the dichroic mirror 106b.

  Further, when measuring the emission amount and emission intensity for each color using light emission of a plurality of colors (that is, when using multicolor light emission), the light emission image pickup unit 106 includes an objective lens 106a and an objective lens 106a as shown in FIG. The CCD camera 106c, the filter wheel 106e, and the imaging lens 106f may be used. Then, the CCD camera 106c may capture a light emission image and a bright field image of the cell 102 projected onto the chip surface of the CCD camera 106c via the filter wheel 106e and the imaging lens 106f. The filter wheel 106e is used when light emitted from the cells 102 is separated by color by exchanging filters, and a light emission amount and light emission intensity are measured for each color using light emission of a plurality of colors.

  Returning to FIG. 1, the image analysis apparatus 110 is specifically a personal computer. As shown in FIG. 4, the image analysis device 110 is roughly divided into a control unit 112, a clock generation unit 114 that measures the system time, a storage unit 116, a communication interface unit 118, and an input / output interface unit. 120, an input device 122, and an output device 124. These units are connected via a bus.

  The storage unit 116 is a storage unit. Specifically, a memory device such as a RAM or a ROM, a fixed disk device such as a hard disk, a flexible disk, an optical disk, or the like can be used. And the memory | storage part 116 memorize | stores the data etc. which were obtained by the process of each part of the control part 112. FIG. The communication interface unit 118 mediates communication between the image analysis device 110 and the CCD camera 106c. That is, the communication interface unit 118 has a function of communicating data with other terminals via a wired or wireless communication line. The input / output interface unit 120 is connected to the input device 122 and the output device 124. Here, in addition to a monitor (including a home television), a speaker or a printer can be used as the output device 124 (hereinafter, the output device 124 may be described as a monitor). In addition to the keyboard, mouse, and microphone, the input device 122 can be a monitor that realizes a pointing device function in cooperation with the mouse.

  The control unit 112 has an internal memory for storing control programs such as an OS (Operating System), programs that define various processing procedures, and necessary data, and performs various processes based on these programs. Run. The control unit 112 is roughly composed of a light emission image capturing instruction unit 112a, a light emission image acquisition unit 112b, an image analysis unit 112c, and an analysis result output unit 112d.

  The light emission image capturing instruction unit 112a instructs the CCD camera 106c to capture a light emitting image and a bright field image via the communication interface unit 118. The luminescent image acquisition unit 112b acquires the luminescent image and bright field image captured by the CCD camera 106c via the communication interface unit 118. Here, the light emission image capturing instruction unit 112a may instruct to repeatedly capture the light emitting image and the bright field image, or may simultaneously perform image capturing for a plurality of different cells 102. Note that the luminescent image acquisition unit 112b may store the captured luminescent image and bright field image in the storage unit 116 together with the time information from the clock generation unit 114.

  In addition, the image analysis unit 112c analyzes the concentration of cyclic GMP based on the emission intensity of the luminescence emitted from the cell 102 based on the emission image acquired by the emission image acquisition unit 112b. Here, the image analysis unit 112c may measure a change in cyclic GMP concentration over time based on a plurality of light emission images repeatedly captured by the light emission image acquisition unit 112b. When a luminescent image is captured, the concentration of cyclic GMP may be analyzed by comparing a plurality of luminescent images for each cell 102.

  The analysis result output unit 112d outputs the analysis result from the image analysis unit 112c to the output device 124. More specifically, the analysis result output unit 112d graphs the time series data of the cyclic GMP concentration based on the emission intensity of the luminescence emitted from the cell 102, which is obtained by the image analysis unit 112c, to the output device 124. indicate.

[Example 1]
Various cyclic GMP sensor proteins were prepared and their characteristics were examined. Specifically, a cyclic GMP sensor protein comprising a cyclic GMP-binding region derived from mouse cyclic GMP-specific phosphodiesterase 5A (PDE5A) and two fragments derived from firefly luciferase was prepared, and responses to various cyclic nucleotides I examined the sex.

(Procedure 1)
Various fragments of luciferase were amplified by PCR (polymerase chain reaction). For the production of the cyclic GMP sensor protein shown in FIG. 5a, the N-terminal luminescent enzyme (NLuc) gene and the C-terminal luminescent enzyme (CLuc) gene were amplified. For the production of the cyclic GMP sensor protein shown in FIG. 5b, the N-terminal luminescent enzyme (cpNLuc) gene and the C-terminal luminescent enzyme (cpCLuc) gene were amplified. The following synthetic oligo DNA was used for amplification.
[Synthetic oligo DNA sequence for NLuc gene preparation]
NLuc_Fw (SEQ ID NO: 1): 5'-TGTGGATCCCAGCCACCCATGAAGATGCCCAA-3 '
NLuc_Rv (SEQ ID NO: 2): 5′-CAGCTCGAGGTCCTTGTCGATGAGAGCGTT-3 ′
[Synthetic oligo DNA sequence for CLuc gene preparation]
CLuc_Fw (SEQ ID NO: 3): 5′-ATCAGATCTGGCTACGTTAACAAACCCCGAG-3 ′
CLuc_Rv (SEQ ID NO: 4): 5′-CTAGAATTCTTACACGGCGATTCTGCCGCC-3 ′
[Synthetic oligo DNA sequence for cpNLuc gene preparation]
cpNLuc_Fw (SEQ ID NO: 5): 5′-ATCAGATCTGAAGATCGCAAAAAACATTAAG-3 ′
cpNLuc_Rv (SEQ ID NO: 6): 5′-CTAGAATTCTTAGTCTCTGTCGATGGAGAGC-3 ′
[Synthetic oligo DNA sequence for cpCLuc gene preparation]
cpCLuc_Fw (SEQ ID NO: 7): 5′-TGTGGATCCCCACCCACATGAGCGGCTACGTTAACAAACCCC-3 ′
cpCLuc_Rv (SEQ ID NO: 8): 5'-CAGCTCCGAGCACGGCGATCTTGCCGCCCTTT-3 '

Moreover, the cGMP binding domain gene of PDE5A was amplified by PCR. Various genes were obtained by using the following primers in combination for amplification.
PDE5A_GAFa_1_Fw (SEQ ID NO: 9): 5′-CCCCCTGAGGTTGATCATGATGGAAGGGGACCAGTGCTCA-3 ′
PDE5A_GAFa_2_Fw (SEQ ID NO: 10): 5′-CCCCTCGAGGTCACAGCATGTGTCACAAA-3 ′
PDE5A_GAFa — 3_Fw (SEQ ID NO: 11): 5′—CTCGAGAAGGAACAAATGCCCACTACACCCC-3 ′
PDE5A_GAFa_1_Rv (SEQ ID NO: 12): 5′-CCCAGATCTCTTGTTCTCCCAGCAGTGAAGTCCTCATAGAG-3 ′
PDE5A_GAFa_2_Rv (SEQ ID NO: 13): 5′-CCCAGATCTCTGAGCATGTGAGAAACAAT-3 ′
PDE5A_GAFa — 3_Rv (SEQ ID NO: 14): 5′-AGATCTGATTAGCTGGCAAGGTCAAGTAA-3 ′
PDE5A_GAFa — 4_Rv (SEQ ID NO: 15): 5′-AGATCTCTTCTTCAGAATGAACTTCCAGTGA-3 ′
PDE5A_GAFa_5_Rv (SEQ ID NO: 16): 5′-AGATCTGGTGCACTTCTGCACCTGCATGAA-3 ′
PDE5A_GAFa — 6_Rv (SEQ ID NO: 17): 5′-AGATCTACAGTCTTCATCCCAAATGAAGAT-3 ′
PDE5A_GAFa — 7_Rv (SEQ ID NO: 18): 5′-AGATCTGGGTTTTCTCACTTCCTCACACTC-3 ′

(Procedure 2)
N-terminal luciferase gene (NLuc), C-terminal luciferase gene (CLuc), mutant N-terminal luciferase gene (cpNLuc) and mutant C-terminal by PCR using pGL4.10 (manufactured by Promega Corporation) as a template The lateral luciferase gene (cpCLuc) was amplified using the primers shown in Table 1 below. The corresponding positions of these genes in the amino acid sequence of luciferase (GL 4.10) are shown in Table 1.

（手順３）
サイクリックＧＭＰ結合領域として使用するために、マウスのサイクリックＧＭＰ特異的ホスホジエステラーゼ５Ａ（ＰＤＥ５Ａ）のｃＤＮＡを鋳型として種々の遺伝子をＰＣＲによって増幅した。図１６に、増幅した遺伝子の名称および使用したプライマーを示す。さらに、増幅した遺伝子の、ＰＤＥ５Ａのアミノ酸配列中の対応する位置、および当該遺伝子がコードする断片のアミノ酸残基の数を示す。なお、ＰＤＥ５ＡにはサイクリックＧＭＰ結合ドメインがＡとＢの２ヶ所存在するが、そのうちＡを含む領域を増幅した。

(Procedure 3)
For use as a cyclic GMP-binding region, various genes were amplified by PCR using the mouse cyclic GMP-specific phosphodiesterase 5A (PDE5A) cDNA as a template. FIG. 16 shows the names of the amplified genes and the primers used. Furthermore, the position of the amplified gene corresponding to the amino acid sequence of PDE5A and the number of amino acid residues of the fragment encoded by the gene are shown. PDE5A has two cyclic GMP binding domains, A and B. Among them, a region containing A was amplified.

(Procedure 4)
Two prokaryotic expression plasmids were prepared as follows. After inserting the NLuc gene obtained in Procedure 2 between the BamHI site and the XhoI site in pRSET2B (manufactured by Invitrogen) which is a prokaryotic expression plasmid, the CLuc gene obtained in Procedure 2 is inserted into the BglII site and the EcoRI site. To insert a prokaryotic expression plasmid (pRSET / GL4_N_C). In addition, after inserting the cpCLuc gene obtained in step 2 between the BamHI site and the XhoI site in pRSET2B, which is a prokaryotic expression plasmid, the cpNLuc gene obtained in step 2 is inserted between the BglII site and the EcoRI site. This was inserted to produce a prokaryotic expression plasmid (pRSET / cpGL4_C_N).

(Procedure 5)
An expression plasmid for cyclic GMP sensor protein was prepared as follows. The 11 PDE5A-derived genes amplified in Procedure 3 were inserted between the XhoI recognition site and the BglII recognition site of the two expression plasmids prepared in Procedure 4, respectively. The names of the 22 types of expression plasmids prepared are shown in the item “Name of the resulting plasmid” in the table of FIG.

(Procedure 6)
Various cyclic GMP sensor proteins were expressed in E. coli and further purified.

The various expression plasmids prepared in Procedure 5 were each transformed into Escherichia coli JM109 (DE3) strain, and cyclic GMP sensor protein was purified using The QIA expressionist (manufactured by Qiagen) according to the manual of the kit. Briefly explaining the purification procedure, the prokaryotic expression plasmid was transformed into E. coli JM109 (DE3) strain and inoculated into 5 mL of LB medium. After overnight culture, the cells were collected by centrifugation, and the collected E. coli cells were resuspended in 500 μL of lysis buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole, pH 8.0), and lysozyme was added (1 mg). / ML) for 60 minutes at 4 ° C. The cells were lysed by gentle agitation using a vortex mixer or the like, centrifuged at 15,000 × g for 10 minutes, and the supernatant was transferred to a new tube. 50 μL of 50% Ni-NTA resin suspension was added and gently mixed at 4 ° C. for 60 minutes. The resin was precipitated by centrifugation at 15,000 × g for 10 seconds, and the resin was washed twice with 500 μL of washing buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 20 mM imidazole, pH 8.0). The protein was eluted twice with 50 μL of elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 250 mM imidazole, pH 8.0) to obtain various cyclic GMP sensor proteins. The obtained 22 types of cyclic GMP sensor proteins are shown in the item of “name of the cyclic GMP sensor protein that can be obtained” in the table of FIG.

(Procedure 7)
The properties of various cyclic GMP sensor proteins were measured.

  The luminescence activity of the protein obtained in Procedure 6 was measured in the absence of cyclic nucleotide, in the presence of cyclic GMP, in the presence of 8-Br-cGMP, or in the presence of cyclic AMP, respectively. To briefly explain the measurement method, 20 μL of phosphate buffer (PBS) was added to 20 μL of the above protein solution. Thereto was added 1 μL of 4 mM cyclic GMP, 8-Br-cGMP or cyclic AMP solution (final concentration 100 μM), and the same amount of PBS was added to the negative control. Furthermore, 40 μL of Bright-Glo (manufactured by Promega) was added, and the amount of luminescence (integration for 5 seconds) was measured using Luminescence JNR-II (manufactured by ATTO).

(result)
The result of the measurement according to procedure 7 is shown in FIG.
In the presence of cyclic GMP, GL4_N_PDE5A_GAFa1_C, GL4_N_PDE5A_GAFa5_C, and GL4_N_PDE5A_GAFa8_C had particularly high light emission amounts. In accordance with these, GL4_N_PDE5A_GAFa7_C, GL4_N_PDE5A_GAFa9_C, and GL4_C_PDE5A_GAFa7_N showed a constant light emission amount.

  Furthermore, these proteins (GL4_N_PDE5A_GAFa1_C, GL4_N_PDE5A_GAFa5_C, GL4_N_PDE5A_GAFa7_C, GL4_N_PDE5A_GAFa8_C, GL4_and luminescence) .

[Example 2]
By the method according to the present invention, the fluctuation of cyclic GMP concentration in HEK293 cells was measured.

(Procedure 1)
Animal cell expression plasmids for various cyclic GMP sensor proteins were prepared as follows.

  Using the restriction enzymes BamHI and EcoRI, a region encoding a cyclic GMP sensor protein was excised from the plasmid for prokaryotic cell expression prepared in Procedure 5 of Example 1, and this was transformed into pcDNA3. 1 (manufactured by Invitrogen) was inserted between the BamHI recognition site and the EcoRI recognition site. The prokaryotic expression plasmids specifically used are four types of pRSET / GL4_N_GAFa1_C, pRSET / GL4_N_GAFa5_C, pRSET / GL4_N_GAFa7_C, and pRSET / cpGL4_C_GAFa7_N, based on these. pcDNA / GL4_N_GAFa5_C, pcDNA / GL4_N_GAFa7_C, and pcDNA / cpGL4_C_GAFa7_N were prepared.

(Procedure 2)
The HEK293 cells used were cultured as follows. HEK293 cells were obtained from ATCC, and cultured in Earle's MEM / medium (GIBCO) supplemented with 10% Fetal Bovine Serum and 1 × Nonesential amino acids in a 5% CO ₂ incubator.

(Procedure 3)
The animal cell expression plasmid prepared in Procedure 1 was introduced into HEK293 cells as follows. Cells were seeded in a 35 mm diameter glass bottom dish at a cell density of 2 × 10 ⁵ / dish, cultured overnight in a 5% CO ₂ incubator, and each animal cell expression plasmid was used with FuGENE HD (Roche). The cells were transfected and cultured overnight in a 5% CO ₂ incubator.

(Procedure 4)
The time lapse photography of the cell produced by the procedure 3 was performed as follows. 2 mM luciferin (manufactured by Promega) was added to the medium and allowed to stand for 1 hour, and set in a light emission microscope LV (LUMINOVIEW) -200 (manufactured by Olympus). As the light emission observation conditions, the magnification of the objective lens is 40 times, the exposure time is 5 to 10 seconds, the binning is 1 × 1, and the EM-CCD camera iXon (manufactured by Andor) is used, and the image analysis apparatus 110 is used. .

(Procedure 5)
Stimulation was given to HEK293 cells during time-lapse photography. Two minutes after the start of time-lapse photography, 8-Br-cGMP (final concentration 100 μM) was added to the culture solution, and then time-lapse photography of the luminescence image was performed.

(Procedure 6)
The emission intensity of the photographed image was analyzed.
An ROI (Region of Interest) was designated in the luminescence image obtained by a series of time-lapse imaging according to procedures 4 and 5. The change over time of the emission intensity in the designated ROI was displayed in a graph.

  FIGS. 7 to 9 show measurement results in cells in which GL4_N_PDE5A_GAFa1_C was expressed. FIG. 7 shows a light emission image before stimulation. 1 to 4 ROIs are designated in the image. On the other hand, FIG. 8 shows a luminescent image obtained by photographing the same position as in FIG. 7 after stimulation. Also in FIG. 8, the ROI is designated as in FIG. Further, FIG. 9 shows a graph showing the change over time of the emission intensity of each designated ROI. The ROIs 1 to 4 specified in FIGS. 7 and 8 correspond to “cell1” to “cell4” in the graph of FIG. 9, respectively. The analysis of the luminescence image was performed using Metamorph software (made by Universal Imaging) that functions as the image analysis unit 112c.

  On the other hand, FIGS. 10 to 12 show measurement results in cells expressing GL4_N_PDE5A_GAFa7_C, and FIGS. 13 to 15 show measurement results in cells expressing cpGL4_C_PDE5A_GAFa7_N. 10 and 13 show the luminescence images before stimulation, FIGS. 11 and 14 show the luminescence images after stimulation, and FIGS. 12 and 15 show graphs of the luminescence intensity of each designated ROI over time. It is.

(result)
8-Br-cGMP is a membrane-permeable cyclic GMP analog, and it is expected that the cyclic GMP inside the cell will increase by adding it to the cell culture medium. The results of reflecting the prediction were obtained by the method according to the present invention using GL4_N_GAFa1_C, GL4_N_GAFa7_C, and cpGL4_C_GAFa7_N, respectively. That is, the amount of luminescence increased after the addition of 8-Br-cGMP. According to the present invention, such a result could be confirmed in a single cell. Moreover, when the change of the emitted light intensity in each cell was seen, it turned out that there is a big dispersion | variation in the response between cells in 8-Br-cGMP stimulation (refer FIG.9, FIG12 and FIG.15).

  Previous studies of intracellular signal transduction mechanisms via cyclic GMP have revealed that the meaning of the signal is different when the intracellular distribution or time course of cyclic GMP concentration changes. In the analysis of changes in the amount of luminescence using a conventional luminometer, it was not possible to analyze the response of individual cells, but only to observe the entire cell population. However, in the method according to the present invention (using LV-200 as an apparatus), changes in the living body can be observed in real time for each cell, and the function of intracellular signals can be studied in detail. .

  As described above, according to the present embodiment, accurate quantitative results from each cell can be obtained without affecting intracellular information transmission. As a result, the change in cyclic GMP concentration in each cell can be obtained. It can be observed over time with good reproducibility. The luciferase gene, luciferin, and the like used in the above-described examples may be other commercially available reagents, or those extracted directly from organisms that generate bioluminescence or the like.

[Other Examples]
In addition to the embodiments shown in the above examples, the present invention can be implemented in various different embodiments within the scope of the technical idea described in the claims.

  For example, in the above-described embodiment, mainly from the N-terminal luminescent enzyme and the C-terminal luminescent enzyme that are separated so that the luminescent enzyme activity is recovered by binding to each other, and the cyclic GMP binding domain of PDE5A However, the present invention is not limited to this, and a cyclic GMP sensor protein disclosed in JP-T-2009-532063 may be used.

  Moreover, according to the description mentioned above, the aspect of polypeptide represented by the following summary is also included by this invention.

[Polypeptide Aspect 1] (Function: Interaction of split luciferase)
A polypeptide comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of a luminescent enzyme, and an amino acid sequence of a cyclic GMP binding protein, wherein the N-terminal fragment and the C-terminal fragment are non-covalently bound A polypeptide that interacts and said non-covalent interaction is tunable.
[Polypeptide Aspect 2] (Function: Action of cyclic GMP binding protein)
The polypeptide according to Aspect 1, wherein the cyclic GMP binding protein detectably changes the non-covalent interaction.
[Polypeptide Aspect 3] (Function: Action of exogenous factor)
The polypeptide according to aspect 1, wherein the non-covalent interaction is enhanced in the presence of an exogenous factor.
[Polypeptide Aspect 4] (Function: Action of cyclic GMP)
The polypeptide according to Aspect 2, wherein the exogenous factor is cyclic GMP.
[Polypeptide Aspect 5] (Function: Luminescence)
4. The polypeptide according to aspect 3, which emits light by the non-covalent interaction.
Below, the claims at the beginning of the filing of the present application are appended as embodiments.
[1]
A cyclic GMP sensor protein comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, and a cyclic GMP binding region, wherein the cyclic GMP binding region comprises the N-terminal fragment and the C-terminal side. A cell production step of producing a cell containing a cyclic GMP sensor protein disposed between the fragments;
An addition step of adding a predetermined luminescent substrate from the outside of the cell prepared in the cell preparation step;
An imaging step of capturing a luminescence image of the cell provided with the predetermined luminescent substrate in the adding step;
An analysis step of analyzing the concentration of the cyclic GMP in the cell based on the luminescence image captured in the imaging step;
An intracellular cyclic GMP analysis method comprising:
[2]
The intracellular cyclic GMP analysis method according to [1], wherein the cyclic GMP binding region is derived from a cyclic GMP-specific phosphodiesterase.
[3]
The cyclic GMP binding region is the amino acid number of mouse cyclic GMP-specific phosphodiesterase 5A.
121st to 330th,
131st to 320th,
131st to 330th,
131st to 343rd, or
131st to 358th
The intracellular cyclic GMP analysis method according to [2], comprising the amino acid sequence of
[4]
The luminescent enzyme is firefly luciferase,
The N-terminal fragment comprises the amino acid sequence of amino acid numbers 1 to 416 of firefly luciferase,
The C-terminal fragment contains the amino acid sequence of amino acid numbers 399 to 550 of firefly luciferase
The intracellular cyclic GMP analysis method according to any one of [1] to [3].
[5]
The intracellular cyclic GMP analysis method according to any one of [1] to [4], wherein the imaging step includes repeatedly capturing the luminescent image.
[6]
The imaging step is performed simultaneously on a plurality of different cells;
The analysis step is performed for each cell;
The intracellular cyclic GMP analysis method according to any one of [1] to [6].
[7]
A cyclic GMP sensor protein comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, and a cyclic GMP binding region, wherein the cyclic GMP binding region comprises the N-terminal fragment and the C-terminal side. Cyclic GMP sensor protein placed between the fragments.
[8]
The cyclic GMP binding region is the amino acid number of mouse cyclic GMP-specific phosphodiesterase 5A.
121st to 330th,
131st to 320th,
131st to 330th,
131st to 343rd, or
131st to 358th
The cyclic GMP sensor protein according to [7], comprising the amino acid sequence of
[9]
The luminescent enzyme is firefly luciferase,
The N-terminal fragment comprises the amino acid sequence of amino acid numbers 1 to 416 of firefly luciferase,
The C-terminal fragment contains the amino acid sequence of amino acid numbers 399 to 550 of firefly luciferase
The cyclic GMP sensor protein according to [7] or [8].
[10]
The cyclic GMP sensor protein according to any one of [7] to [9], comprising the amino acid sequence represented by SEQ ID NO: 31, 35, 37, 48, 38 or 39.

  As described above in detail, the cyclic GMP analysis method according to the present invention can be suitably used in various fields such as biotechnology, pharmaceuticals, and medicine.

100 Luminescence observation system
103 container (petri dish)
104 stages
106 Luminous image pickup unit
106a Objective lens (for light emission observation)
106b Dichroic mirror
106c CCD camera
106d Split image unit
106e Filter wheel
106f Imaging lens
110 Image analyzer
112 Control unit
112a Luminous image capturing instruction unit
112b Luminescent image acquisition unit
112c Image analysis unit
112d Analysis result output part
114 Clock generator
116 storage unit
118 Communication interface
120 I / O interface section
122 Input device
124 Output device

Claims (4)

A cell production step of producing a cell containing a cyclic GMP sensor protein comprising the amino acid sequence shown in SEQ ID NO: 31, 35, 37, 48, 38 or 39 ;
An addition step of adding a predetermined luminescent substrate from the outside of the cell prepared in the cell preparation step;
An imaging step of capturing a luminescence image of the cell provided with the predetermined luminescent substrate in the adding step;
An intracellular cyclic GMP analysis method including an analysis step of analyzing the concentration of the cyclic GMP in the cell for each cell based on the luminescence image captured in the imaging step. The intracellular cyclic GMP analysis method according to claim 1 , wherein the imaging step includes repeatedly capturing the luminescent image. The imaging step is performed simultaneously on a plurality of different cells;
The analysis step is performed for each cell;
The intracellular cyclic GMP analysis method according to claim 1 or 2 . A cyclic GMP sensor protein comprising the amino acid sequence shown in SEQ ID NO: 31, 35, 37, 48, 38 or 39.

Images