JP5893241B2 - Calcium analysis method - Google Patents

Description

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

  Many hormones and neurotransmitters interact with 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.

  Calcium, which is one of intracellular second messengers, acts as a signal transduction molecule for various biochemical reaction processes in vivo. To date, calcium regulates various physiological processes such as muscle cell relaxation, photoreception-transmission in the retina, bone growth, neuronal activation, and intracellular signaling from neurotransmitters and hormone receptors. It has become clear that Therefore, if calcium localization in living cells is known in real time, not only the mechanisms of action such as the circulatory system, digestive system, retina, olfaction, and central nerves at the cellular and tissue levels are elucidated, but also intracellular It is expected to obtain knowledge about drugs that regulate calcium concentration in the body.

  In order to observe the calcium concentration in a living cell in real time, molecular imaging of calcium is the most effective, and many analytical techniques for calcium have been proposed and developed.

  For example, Non-Patent Document 1 discloses a fusion protein in which cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) are fused to calmodulin that is a calcium binding protein and M13 peptide that binds to calmodulin bound to calcium ions. Calcium analysis method based on fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) is disclosed. The fusion protein used in this method has calmodulin and M13 peptide at the center and YFP and CFP at both ends. In the absence of calcium, YFP and CFP are separated from each other and FRET does not occur. Therefore, even when CFP is excited, YFP fluorescence is not observed. At this time, when intracellular calcium increases, calmodulin binds to calcium, and M13 binds to them to change the three-dimensional structure of the fusion protein. When CFP is excited in this state, FRET is generated, and fluorescence of YFP as an acceptor is observed. It has been clarified that the change in calcium concentration can be observed by observing the FRET signal.

Patent Document 1 discloses a method for quantitatively analyzing intracellular calcium concentration and gene expression level using calcium-sensitive photoprotein and calcium non-responsive Renilla luciferase. In this method, reporters derived from aequorin and calcium non-responsive Renilla luciferase, which are calcium-sensitive photoproteins, are expressed in cells.
It has been shown that increase in intracellular calcium can be detected by aequorin or calcium-sensitive photoprotein, and then the increase or decrease in gene expression can be measured by a reporter assay without the addition of a new substrate.

  Non-Patent Document 2 discloses a method of observation using a fusion protein composed of calmodulin, M13 peptide, and split Renilla luciferase. In the fusion protein used in this method, calmodulin and M13 peptide are arranged at the center, and a fragment obtained by dividing Renilla luciferase is arranged at both ends. In the absence of calcium, luminescence cannot be observed because the fragments of Renilla luciferase are separated from each other and have no luminescence activity. At this time, when intracellular calcium increases, calmodulin binds to calcium, and M13 binds to them to change the three-dimensional structure of the fusion protein. In this state, the divided Renilla luciferase is reconstituted to recover the luminescence activity, and the luminescence can be observed. It has been clarified that the change in calcium concentration can be observed by observing this luminescent signal.

  Patent Document 2 discloses a method of observation using a fusion protein composed of calmodulin, optionally M13 peptide, and split Gaussia luciferase. The fusion protein used in this method has calmodulin and optionally M13 peptide at the center and a fragment of Gaussia luciferase at both ends. As the intracellular calcium increases, calmodulin and optionally M13 peptide The three-dimensional structure changes by binding in a calcium-dependent manner. This change allows the resolved Gaussia luciferase to dissociate or reconstitute and observe the disappearance or enhancement of luminescence. It has been clarified that the change in calcium concentration can be observed by observing this luminescent signal.

  Patent Document 3 discloses a method for quantitative analysis of intracellular calcium concentration using a fusion protein composed of calmodulin and divided firefly luciferase. In the fusion protein used in this method, the divided firefly luciferase fragments are circularly permuted with respect to the arrangement in the original firefly luciferase. When this fusion protein is expressed in cells and luciferin serving as a luciferase substrate is added, luminescence from the cells can be measured. At this time, when intracellular calcium increases, the three-dimensional structure of calmodulin changes, and the luminescence of luciferase disappears or is enhanced. It has been clarified that a change in calcium concentration can be detected by detecting this luminescent signal.

International Publication No. 2007/139080 JP 2009-153399 A JP 2009-532063 A

Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY. “Fluorescent indicators for Ca2 + based on green fluorescent proteins and calmodulin.” Nature, vol. 388 (6665), pp. 882-887, 1997 Kaihara A, Umezawa Y, Furukawa T .; “Bioluminous indicators for Ca2 + based on split renilla luciferase complementation in living cells.” Analytical Sciences, Vol. 24, pp. 1405-1408, 2008

  However, the conventional technique using a fluorescent protein has a problem that a signal / noise ratio is low because cells emit autofluorescence, a problem that a range of usable measurement objects is narrow because a dynamic range is narrow, and the like.

  In addition, the conventional technique using the conventional calcium-sensitive photoprotein is measured only with a luminometer or photon counting, so it is not optimized for single cell measurement. Since it is decomposed, it is difficult to observe light emission corresponding to a long-time measurement or many times of stimulation, and there is a problem that the range of available measurement methods is narrow.

  In addition, the conventional technology using conventional split Renilla luciferase or Gaussia luciferase, calcium-binding protein calmodulin and calmodulin bound to calcium ion is measured only by luminometer or photon counting. Therefore, it has not been optimized for measurement in one cell, and furthermore, since the reconstituted luciferase is difficult to dissociate, there is a problem that the correlation between the luminescence signal and the calcium concentration is poor.

  In addition, the conventional technique using a fusion protein of a circular permutation luciferase and the calcium-binding protein calmodulin gene is measured only with a luminometer or photon counting, so it is not optimized for single cell measurement. However, there is a problem that the range of available measurement methods is narrow.

  The present invention has been made in view of the above problems, and can obtain an accurate quantitative result from each cell without affecting intracellular information transmission. As a result, calcium in each cell can be obtained. It is an object of the present invention to provide a calcium analysis method capable of observing changes in concentration over time with good reproducibility.

  According to an embodiment of the present invention, a calcium sensor protein comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, a calcium binding region, and an interaction region capable of reversibly binding to or dissociating from the calcium binding region. A cell production step for producing a cell containing a calcium sensor protein in which the calcium binding region and the interaction region are arranged between the C-terminal fragment and the N-terminal fragment; and the cell production step An addition step of adding a predetermined luminescent substrate from outside the cell to the cells prepared in Step 1, 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 imaging step An intracellular calculus comprising an analysis step of analyzing the concentration of the calcium in the cell based on the captured luminescence image Analysis method is provided.

  In addition, according to an embodiment of the present invention, calcium containing an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, a calcium binding region, and an interaction region capable of reversibly binding to or dissociating from the calcium binding region A sensor protein, wherein the calcium-binding region and the interaction region are disposed between the C-terminal fragment and the N-terminal fragment, and the N-terminal fragment and the C-terminal fragment are inherent in the fermentation enzyme. Calcium sensor proteins are provided that are circularly permuted to configuration.

  According to the embodiments of the present invention, it is possible to obtain accurate quantitative results from individual cells regarding intracellular calcium, and as a result, it is possible to observe the intracellular calcium concentration in individual cells with good reproducibility. The effect that it can be obtained.

  Moreover, according to the embodiment in which the calcium binding region of the present invention is derived from calmodulin, an effect of binding specifically to calcium can be obtained.

  Moreover, according to the embodiment in which the interaction region of the present invention is the M13 peptide, an effect of binding to calmodulin in a calcium-dependent manner is obtained.

  Moreover, the luminescent enzyme of the present invention 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 amino acid numbers 399 to 550 of firefly luciferase. According to the embodiment including the amino acid sequence, light is emitted only when the N-terminal fragment and the C-terminal fragment of the luminescent enzyme are reconstituted, so that an effect that the signal / noise ratio is large is obtained.

  Further, the luminescent enzyme of the present invention is Renilla luciferase, the N-terminal fragment contains the amino acid sequence from amino acid number 1 to 91 of Renilla luciferase, and the C-terminal fragment from amino acid number 92 of Renilla luciferase. According to the embodiment including the 311th amino acid sequence, light is emitted only when the N-terminal fragment and the C-terminal fragment of the luminescent enzyme are reconstituted, so that an effect that the signal / noise ratio is large is obtained.

  In addition, according to the embodiment in which the N-terminal fragment and the C-terminal fragment of the present invention are circularly permuted with respect to the original arrangement in the fermentation enzyme, since the background noise when dissociated is low, the signal / noise ratio The effect is large.

  Further, according to the embodiment in which the imaging step of the present invention includes repeatedly capturing a luminescent image, the luminescent image is repeatedly captured in the imaging step, so that an effect of being able to perform spatiotemporal measurement in the same cell is obtained. .

  Further, according to the embodiment in which the imaging process of the present invention is performed simultaneously on a plurality of different cells and the analysis process is performed for each cell, the imaging process is performed simultaneously on a plurality of different cells, Since a plurality of luminescent images are collated for each cell, an effect that the change in calcium concentration in each cell can be analyzed in time and space can be obtained.

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. 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. 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 calcium using the split luciferase assay method. It is a figure which shows the luminescence image before 5HT stimulation in HEK293 cell which introduce | transduced 5HT2A receptor and cpGL4_C_CaM-M13_N. It is a figure which shows the luminescence image after 5HT stimulation in HEK293 cell which introduce | transduced 5HT2A receptor and cpGL4_C_CaM-M13_N. It is the figure which showed the time-dependent change of the luminescence intensity of cpGL4_C_CaM-M13_N by 5HT stimulation in the selected cell. It is a figure which shows the luminescence image before 5HT stimulation in HEK293 cell which introduce | transduced 5HT2A receptor and cphRL_C_CaM-M13_N. It is a figure which shows the luminescent image after 5HT stimulation in HEK293 cell which introduce | transduced 5HT2A receptor and cphRL_C_CaM-M13_N. It is the figure which showed the time-dependent change of the emitted light intensity of cphRL_C_CaM-M13_N by 5HT stimulation in the selected cell.

[Method]
The method according to the present invention will be described below.
The present invention is a calcium sensor protein comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, a calcium binding region, and an interaction region capable of reversibly binding to or dissociating from the calcium binding region, A cell preparation step for preparing a cell containing a calcium sensor protein arranged between the C-terminal fragment and the N-terminal fragment for the calcium binding region and the interaction region; and the cell prepared in the cell preparation step An addition step of adding a predetermined luminescent substrate from the outside of the cell, an imaging step of capturing a luminescent image of the cell to which the predetermined luminescent substrate is given in the addition step, and the luminescent image captured in the imaging step And an analysis step of analyzing the concentration of the calcium in the cell based on the method .

  Briefly, the present invention relates to a method of introducing a calcium sensor protein into a target cell, inducing luminescence by giving a luminescent substrate, imaging the state, and specifying a calcium concentration based on the captured image. .

  According to the present invention, the state of intracellular calcium can be analyzed. In particular, the intracellular calcium concentration can be quantitatively measured. 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, it is possible to analyze changes in calcium concentration with respect to stimulation and temporal changes. Differences between cells can be analyzed by measuring each cell.

  In the cell production process, cells containing the calcium sensor protein are produced. The calcium sensor protein includes two polypeptide fragments of a luminescent enzyme, a polypeptide fragment that can bind to calcium (calcium binding region), and a polypeptide fragment that can reversibly bind to or dissociate from the calcium binding region (interaction region). A protein that is optionally fused with other polypeptide fragments, wherein the activity of the luminescent enzyme disappears or recovers depending on the presence of calcium. Details of the calcium 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 calcium 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, It can select suitably. For example, by adding to the culture solution, the luminescent substrate can be taken into the cell 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 calcium 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 calcium sensor protein may be introduced into a cell, and light from the specific protein may be detected simultaneously with light derived from the calcium sensor protein. . An apparatus used for imaging will be described later.

  In the analysis step, the intracellular calcium concentration and the like are analyzed 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 treatment, the intracellular calcium concentration is specified. Further, the specification of the concentration may be a rough specification such as whether or not calcium is present in the sample or whether or not it is within a set concentration range, without specifying a detailed numerical value. An apparatus used for analysis will be described later.

  The present invention also relates to the above-described intracellular calcium 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 calcium concentration according to time or according to stimulation. Further, by acquiring a bright field image at the same time, it is possible to know the relationship between a change in calcium concentration and a change in cell morphology. Moreover, the relationship between the protein and calcium can be known by expressing the protein with the probe and observing the state of the probe at the same time.

  The present invention also relates to an intracellular calcium 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 methods, changes in calcium concentration in individual cells can be analyzed spatiotemporally. For example, it can be used to compare calcium concentrations between cells placed under the same conditions.

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

  The calcium sensor protein according to the present invention comprises an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of a luminescent enzyme, a calcium binding region and an interaction region capable of reversibly binding to or dissociating from the calcium binding region, and calcium binding The region and the interaction region are located between the N-terminal fragment and the C-terminal fragment. The protein loses or recovers the activity of the luminescent enzyme depending on the presence of calcium.

  The calcium binding region means a peptide that can reversibly bind and dissociate with calcium ions in cells. A specific example of the calcium binding region is, for example, calmodulin (CaM) or a fragment thereof. On the other hand, the interaction region means a peptide capable of interacting with the calcium binding region. In particular, the interaction region can bind to the calcium binding region bound to the calcium ion, and can dissociate from the calcium binding region from which the calcium ion is dissociated. A specific example of the interaction region is, for example, M13 peptide.

  As the calcium-binding region and interaction region, a peptide in which calmodulin (CaM), which is a calcium-binding protein, and M13, which binds to calmodulin bound to calcium ions, are connected in series can be used (Non-patent Document 1). In Non-Patent Document 1, a calcium sensor protein having a structure in which CaM and M13 are sandwiched between two types of fluorescent proteins is prepared using a linked peptide connected in this series, and this fluorescent sensor protein is referred to as “chameleon”. It is called "Protein (hereinafter referred to as Chameleon)". In particular, as a calcium binding region and an interaction region, a peptide corresponding to the amino acid sequence from chameleon 230 to 406, a peptide corresponding to 230 to 396, a peptide corresponding to 230 to 401, 230 to 411 The peptide corresponding to the th or the peptide corresponding to the 230 th to 416 th can be used. Peptides with these specific sequences can be used for the calcium sensor protein according to the present invention as long as they do not lose their functions, for example, even those containing mutations for improving sensitivity. it can.

  A luminescent enzyme means an enzyme that catalyzes a reaction involving luminescence. The luminescent enzyme is contained as an N-terminal fragment and a C-terminal fragment in the calcium sensor protein. 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.

  For example, luciferase can be used as the luminescent enzyme. Luciferases include firefly-derived, Renilla-derived, and bacteria-derived luciferases, and it is particularly preferable to use firefly-derived luciferase or Renilla-derived luciferase. The luminescent substrate is selected according to the luminescent enzyme. When firefly luciferase is used as the luminescent enzyme, luciferin can be used as the luminescent substrate. Moreover, when using as a Renilla luciferase luminescent enzyme, coelenterazine can be used as a luminescent substrate. In particular, a peptide fragment corresponding to the 1st to 416th amino acid sequences of firefly luciferase is used as an N-terminal fragment, and a peptide fragment corresponding to the 399th to 550th amino acid sequences of firefly luciferase is used as a C-terminal fragment. can do. Alternatively, a peptide fragment corresponding to the first to 91st amino acid sequence of Renilla luciferase is used as the N-terminal fragment, and a peptide fragment corresponding to the 92nd to 311th amino acid sequence of Renilla luciferase is used as the C-terminal fragment. Can be used. Peptides with these specific sequences can be used for the calcium sensor protein according to the present invention as long as they do not lose their functions, for example, even if they contain mutations for improving activity. it can.

  In the calcium sensor protein, the calcium binding region and the interaction region are arranged between the C-terminal fragment and the N-terminal fragment. Either the calcium binding region or the interaction region may be located on the N-terminal side of the calcium sensor protein, but when using the above-mentioned chameleon protein-derived CaM-M13, the calcium binding region is located on the N-terminal side of the calcium sensor protein. A certain CaM is located, and M13 which is an interaction region is located on the C-terminal side. On the other hand, either the C-terminal fragment or the N-terminal fragment of the luminescent enzyme may be located on the N-terminal side of the calcium sensor protein. For example, the N-terminal fragment of the luminescent enzyme may be disposed on the N-terminal side of the calcium sensor protein, and the C-terminal fragment of the luminescent enzyme may be disposed on the C-terminal side of the calcium sensor protein. In contrast, the N-terminal fragment and the C-terminal fragment may be circularly permuted with respect to the original arrangement in the fermentation enzyme. That is, the C-terminal fragment of the luminescent enzyme may be arranged on the N-terminal side of the calcium sensor protein, and the N-terminal fragment of the luminescent enzyme may be arranged on the C-terminal side of the calcium sensor protein.

  Accordingly, the calcium center protein has, for example, a structure in which the C-terminal fragment of the luminescent enzyme, the calcium binding region, the interaction region, and the N-terminal fragment of the luminescent enzyme are arranged in this order from the N-terminal side. As a specific example of this, the calcium sensor protein may have the amino acid sequence shown in SEQ ID NO: 21. From the N-terminal side, this protein consists of a C-terminal fragment of firefly luciferase (399th to 550th), a CaM-M13 fragment of chameleon protein (230th to 406th) and an N-terminal fragment of firefly luciferase (first) 416th from the top) and is referred to as “cpGL4_C_CaM-M13_N” in this application (see FIG. 5a). The calcium sensor protein may have the amino acid sequence shown in SEQ ID NO: 22. From the N-terminal side of this protein, the C-terminal fragment of Renilla luciferase (92nd to 311th), the CaM-M13 fragment of Chameleon protein (230th to 406th) and the N-terminal fragment of Renilla luciferase ( 1 to 91), which is referred to as “cphRL_C_CaM-M13_N” in the present application (see FIG. 5b).

  The calcium sensor protein can acquire or lose activity as a luminescent enzyme depending on the presence of calcium. Although the C-terminal side fragment and the N-terminal side fragment of the luminescent enzyme contained in the calcium sensor protein have lost the activity as the luminescent enzyme, respectively, the C-terminal side fragment and the N-terminal side fragment depending on the structure of the calcium sensor protein The luminescent activity can be recovered by the close binding of. The structure of the calcium sensor protein is regulated by the calcium binding region and the interaction region. For example, as shown in FIG. 5a, in the case of cpGL4_C_CaM-M13_N, when no calcium ion is present, the C-terminal fragment and the N-terminal fragment are bound close to each other and show activity as a luminescent enzyme. When calcium ions are present, calcium ions bind to CaM, and further M13 binds thereto, thereby changing the structure of the entire sensor protein and losing the activity as a luminescent enzyme. On the other hand, in the case of cphRL_C_CaM-M13_N shown in FIG. 5b, when no calcium ion is present, the C-terminal fragment and the N-terminal fragment cannot bind to each other and do not exhibit activity as a luminescent enzyme. When present, the structure of the sensor protein is changed by the action of CaM and M13, and the C-terminal side fragment and the N-terminal side fragment bind to each other in a moderately close manner, thus showing activity as a luminescent enzyme. Based on such a mechanism, the presence or absence of calcium ions can be detected as the presence or absence of luminescence by a luminescent enzyme by using a calcium sensor protein.

  In addition, there is no particular limitation on the mode of “bonding” between the C-terminal fragment and the N-terminal fragment of the luminescent enzyme, and chemical bonds such as covalent bonds or non-covalent bonds may be used. In particular, it is preferable that the N-terminal side fragment and the C-terminal side fragment are in “close” or “contact” to such an extent that the original function of the luminescent enzyme can be achieved. 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”.

  As described above, the calcium sensor protein according to the present invention loses or recovers the activity of the luminescent enzyme depending on the presence of calcium. In addition, the method according to the present invention utilizes such characteristics of the protein. Conventional technologies using intermolecular energy resonance (FRET, BRET, etc.) have a narrow dynamic range due to the low efficiency of energy resonance, and the presence / absence of calcium depending on the difference in the detected wavelength. Is analyzed. On the other hand, in the present invention, since a simple reaction by a luminescent enzyme such as luciferase is used, the dynamic range is wide, imaging is possible, and the presence / absence of calcium corresponds to the occurrence / non-occurrence of luminescence. A detection result with a high signal / noise ratio can be obtained.

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

  The nucleic acid includes a gene encoding the calcium 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 calcium sensor protein, a sequence included in a general expression vector such as a sequence for controlling expression, a sequence of a marker gene, and the like may be included. The expression vector can be used in the cell preparation step of the intracellular calcium analysis method according to the present invention. That is, the expression vector can be introduced into a cell to express a calcium sensor protein and used for detection of calcium.

[apparatus]
Next, the luminescence observation system 100 used in the calcium 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 signal / noise 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 luminescently labeled so as to emit light depending on the calcium concentration. In addition, as a protein that emits light depending on the calcium concentration, a calcium binding region (calmodulin) and an interaction region that can reversibly bind to or dissociate from the calcium binding region between the C-terminal fragment and the N-terminal fragment of the luminescent enzyme In addition to the fusion protein into which (M13) is inserted, for example, a fusion protein disclosed in Patent Document 2 or Patent Document 3 may be used. 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.

  Further, the image analysis unit 112c analyzes the concentration of calcium based on the emission intensity of the luminescence emitted from the cell 102 based on the luminescence image acquired by the luminescence image acquisition unit 112b. Here, the image analysis unit 112c may measure a variation in calcium concentration over time based on a plurality of light emission images repeatedly captured by the light emission image acquisition unit 112b, and the light emission images for a plurality of different cells 102 simultaneously. 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 calcium concentration based on the emission intensity of the luminescence emitted from the cell 102 obtained by the image analysis unit 112c and displays the graph on the output device 124. .

  A calcium sensor protein using firefly luciferase was prepared and its function was examined.

[Preparation 1]
As preparations, the N-terminal fragment gene and the C-terminal fragment gene of firefly luciferase were cloned, the CaM and M13 genes were cloned, and a calcium sensor protein expression plasmid was prepared.

(Procedure 1)
Synthetic oligo DNA (deoxyribonucleic acid) used for PCR (polymerase chain reaction) for cloning each fragment of firefly luciferase was prepared with the sequence shown below. The prepared oligo DNA has an N-terminal fragment (NLuc) gene and a C-terminal fragment (CLuc) gene for placement in the sensor protein in the original order, and a circular permutation for placement in the sensor protein. N-terminal fragment (cpNLuc) gene and C-terminal fragment (cpCLuc) gene.
[Synthetic oligo DNA sequence for NLuc gene preparation]
NLuc_Fw (SEQ ID NO: 1): 5'-TGTGGATCCCAGCCACCCATGAGATAGGCCAA-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′-ATCAGATCTGGCTACGTTAACAACCCCGAG-3 ′
CLuc_Rv (SEQ ID NO: 4): 5′-CTAGAATTCTTACACCGGCGATCTGGCCGCC-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 '

In addition, synthetic oligo DNA used for PCR for cloning of calmodulin and M13 was prepared with the sequences shown below.
[Synthetic oligo DNA sequence for preparing CaM-M13 gene]
CaM-M13_Fw (SEQ ID NO: 9): 5′-GCCCTCGAGCATGACCAACTGACAGAAGAG-3 ′
CaM-M13_Rv (SEQ ID NO: 10): 5'-CATGGATCCCCAGTGCCCCGAGCTGGAGAT-3 '
CaM-M13d10_Rv (SEQ ID NO: 11): 5′-CATGGATCCCCGGTTGGCAGCCGCTGACGGC-3 ′
CaM-M13d05_Rv (SEQ ID NO: 12): 5′-CATGGATCCGAGATCTTCTTTGAACCGGTT-3 ′
CaM-M13a05_Rv (SEQ ID NO: 13): 5′-CATGGATCCGCTCCATCATAGCTCCAGTG-3 ′
CaM-M13a10_Rv (SEQ ID NO: 14): 5′-CATGGATCCCCAGCTCCCTCCCCCTGCTCAC-3 ′

(Procedure 2)
Each fragment of firefly luciferase was cloned by PCR using the above oligo DNA. PGL4.10 (manufactured by Promega Corporation) was used as a template. Using the oligo DNAs of SEQ ID NOs: 1 and 2 as primers, the N-terminal fragment gene (NLuc: including amino acids 1 to 416 of GL 4.10 gene) is amplified, and the oligo DNAs of SEQ ID NOs: 3 and 4 are used. C-terminal fragment gene (CLuc: 399th to 550th amino acids of GL4.10 gene) are amplified, and N-terminal fragment for circular permutation sensor protein using oligo DNAs of SEQ ID NOs: 5 and 6 A gene (including the 1st to 416th amino acids of cpNLuc: GL4.10 gene) is amplified, and the C-terminal fragment gene for circular permutation sensor protein (cpCLuc: GL4) is used with the oligo DNAs of SEQ ID NOs: 7 and 8 .10 genes (including amino acids 399 to 550) were amplified.

(Procedure 3)
CaM and M13 were cloned by PCR using the above oligo DNA. The chameleon gene (YC2.1) cDNA was used as a template (Non-patent Document 1). Using the oligo DNAs of SEQ ID NOs: 9 and 10 as primers, the CaM-M13 gene (including the region corresponding to the 230th to 406th amino acid sequences of the chameleon gene), and the oligo DNAs of SEQ ID NOs: 9 and 11 as primers The CaM-M13d10 gene (including the region corresponding to the 230th to 396th amino acid sequence of the chameleon gene) and the oligo DNA of SEQ ID NOs: 9 and 12 were used as primers, and the CaM-M13d05 gene (230th to 401th of the chameleon gene). A region corresponding to the amino acid sequence of No. 9), CaM-M13a05 gene (including a region corresponding to the 230th to 411st amino acid sequences of the chameleon gene) and sequences using the oligo DNAs of SEQ ID NOs: 9 and 13 as primers Issue 9 and 14 of the CaM-M13a10 gene using the oligo DNA as a primer (containing the region corresponding to the 416 amino acid sequence from 230 th chameleon gene) were amplified respectively.

(Procedure 4)
Each gene amplified as described above was inserted into an expression plasmid to prepare a calcium sensor protein expression plasmid. Specifically, an NLuc gene and a CLuc gene are used as a luminescent enzyme fragment between a BamHI site and an EcoRI site of pcDNA3.1 (manufactured by Invitrogen), which is a plasmid for animal cell expression, as a calcium binding region and an interaction region. CaM-M13 gene, CaM-M13d10 gene, CaM-M13d05 gene, CaM-M13a05 gene or CaM-M13a10 gene are inserted, respectively, and plasmid pcDNA / GL4_N_CaM-M13_C, pcDNA / GL4_N_CaM-M13d10_C, pcDNA / GL4_N_CaC, GL4_N_CaM-M13a05_C and pcDNA / GL4_N_CaM-M13a10_C were prepared. Further, between the BamHI site and EcoRI site of pcDNA3.1 (manufactured by Invitrogen), cpNLuc gene and cpCLuc gene are used as luminescent enzyme fragments, CaM-M13 gene, CaM-M13d10 gene are used as calcium binding region and interaction region, CaM-M13d05 gene, CaM-M13a05 gene or CaM-M13a10 gene is inserted, respectively, and plasmids pcDNA / cpGL4_C_CaM-M13_N, pcDNA / cpGL4_C_CaM-M13d10_N, pcDNA / cpGL4_C_CaM-M13d05_N, pcDNA / Mc_c13 Produced.

[Preparation 2]
In order to measure the function of the calcium sensor protein, an experimental system in which calcium ions are allowed to flow by stimulating cells with serotonin is used. Therefore, the gene of 5HT2A receptor (SEQ ID NO: 23), which is a serotonin receptor, was cloned.

(Procedure 1)
Synthetic oligo DNA used for PCR was prepared with the sequence shown below.
[Synthetic oligo DNA sequence for preparing human 5HT2A receptor gene]
HU — 5HT2AR_Fw (SEQ ID NO: 15): 5′-GCCACATGGGAATTTCTTGTGAAGAAAAAT-3 ′
HU — 5HT2AR_Rv (SEQ ID NO: 16): 5′-TCACACACAGCTCACCTTTTCATTCACTCC-3 ′

(Procedure 2)
Using the above oligo DNA as a primer and a human brain cDNA library (manufactured by Takara Bio Inc.) as a template, 5HT2A receptor gene (including a region corresponding to the full length of human 5HT2A receptor gene) is amplified by PCR. did.

(Procedure 3)
After subcloning the 5HT2A receptor gene amplified by PCR into the pBluescript II vector, the sequence of the prepared DNA was confirmed by DNA sequencing.

(Procedure 4)
The 5HT2A receptor gene amplified as described in Procedure 2 was inserted into a mammalian cell expression vector pcDNA3.1 (+) (manufactured by Invitrogen Corp.) to prepare a human 5HT2A receptor expression plasmid pcDNA / 5HT2AR.

[Experiment]
Using the calcium sensor protein expression plasmid and the human 5HT2A receptor expression plasmid prepared as described above, luminescence imaging of calcium concentration fluctuations in HEK293 cells was performed.

(Procedure 1)
HEK293 cells obtained from ATCC were 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 2)
Each expression plasmid was introduced into the cells as follows. Cells were seeded in a 35 mm diameter glass bottom dish at a cell density of 2 × 10 ⁵ / dish and cultured overnight in a 5% CO ₂ incubator, and then a plasmid pcDNA / 5HT2AR for expressing human 5HT2A receptor and a plasmid for expressing calcium sensor protein pcDNA / GL4_N_CaM-M13_C, pcDNA / GL4_N_CaM-M13d10_C, pcDNA / GL4_N_CaM-M13d05_C, pcDNA / GL4_N_CaM-M13a05_C, pcDNA / cpGL4_C_CaM-M13_N, pcDNA / cpGL4_C_CaM-M13d10_N, the pcDNA / cpGL4_C_CaM-M13d05_N or pcDNA / cpGL4_C_CaM-M13a05_N, FuGENE H Transfection was performed using D (Roche) and the cells were cultured overnight in a 5% CO ₂ incubator.

(Procedure 3)
The emission image was taken as follows. 2 mM luciferin (manufactured by Promega) was added to the medium and allowed to stand for 1 hour, set in a light emission microscope LV (LUMINOWVIEW) -200 (manufactured by Olympus), and time-lapse photography of the luminescence images was performed at 12-second intervals. The personal computer configured as the image analysis apparatus 110 using the EM-CCD camera iXon (manufactured by Andor) with the magnification of the objective lens being 40 times, the exposure time being 5 to 10 seconds, the binning being 1 × 1, as the conditions for the light emission observation Incorporated.

(Procedure 4)
Two minutes after the start of time-lapse photography, stimulation was performed with serotonin 5HT (final concentration 10 μM), and then time-lapse photography of the luminescence image was performed. This stimulation causes calcium ions to flow into the cell.

(Procedure 5)
A plurality of ROIs (Region of Interest) are designated for each light emission image taken in step 3 (FIG. 6), and a plurality of ROIs are designated for each light emission image taken in step 4 ( FIG. 7). Furthermore, the emission intensity of each designated ROI was measured based on each emission image, and the change over time in the emission intensity was displayed in a graph (FIG. 8). 6 to 8 show the results when cpGL4_C_CaM-M13d10_N is expressed in cells. The analysis of the luminescence image was performed using MetaMorph software (manufactured by Universal Imaging) that functions as the image analysis unit 112c.

[result]
As shown in FIG. 6-8, when the calcium sensor protein cpGL4_C_CaM-M13_N (SEQ ID NO: 21) using a circular permutation type firefly luciferase gene is used, a change in luminescence intensity is detected before and after the serotonin 5HT stimulation. I was able to. That is, the luminescence intensity observed before stimulation (FIG. 6) decreased after stimulation (FIG. 7). From the graph, it can be seen that the fluorescence intensity greatly decreases immediately after 5HT stimulation (2-3 minutes on the horizontal axis) (FIG. 8). In addition, after 5 minutes, it was observed that the emission intensity increased and decreased, which is considered to reflect the regulation of the calcium concentration by the cells. Thus, it was found that by using cpGL4_C_CaM-M13_N, the calcium response to the stimulus can be detected at a single cell (single cell) level as a decrease in luminescence intensity.

  On the other hand, when pcDNA / GL4_N_CaM-M13_C, pcDNA / GL4_N_CaM-M13d10_C, pcDNA / GL4_N_CaM-M13d05_C or pcDNA / GL4_N_CaM-M13a05_C was used, there was no change in the luminescence intensity when the cells were stimulated with 5HT, and p was not observed. When cpGL4_C_CaM-M13d10_N, pcDNA / cpGL4_C_CaM-M13d05_N, and pcDNA / cpGL4_C_CaM-M13a05_N were used, the emission was weak and image acquisition was not possible.

  Moreover, when the change of the emitted light intensity in each cell was seen, it turned out that there is big dispersion | variation in the response between cells in 5HT stimulation (FIG. 8). Previous studies on intracellular signal transduction mechanisms via calcium have revealed that the meaning of the signal differs depending on the intracellular distribution or time course of changes in calcium concentration. 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 case of the method using the calcium sensor protein according to the present invention, it was found that 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.

  A calcium sensor protein using Renilla luciferase was prepared and its function was examined.

[Preparation 1]
As preparations, cloning of the N-terminal fragment gene and C-terminal fragment gene of Renilla luciferase, cloning of the CaM and M13 genes, and preparation of a calcium sensor protein expression plasmid were performed.

(Procedure 1)
Synthetic oligo DNAs used for PCR for the preparation of circularly permuted Renilla luciferase N-terminal fragment (cpRNLuc) gene and C-terminal fragment (cpRCLuc) gene were prepared with the sequences shown below.
[Synthetic oligo DNA sequence for cpRNLuc gene preparation]
cpRNLuc_Fw (SEQ ID NO: 17): 5′-ATCAGATCTGCTTCCAAGGTGTACACCCC-3 ′
cpRNLuc_Rv (SEQ ID NO: 18): 5′-CTAGAATTCTTATGAGCCATTCCCGCTCTT-3 ′
[Synthetic oligo DNA sequence for preparing cpRCluc gene]
cpRCLUc_Fw (SEQ ID NO: 19): 5′-TGTGGATCCCAGCCACCATGTATCGCCTCTCTGGATCACTAC-3 ′
cpRCLUc_Rv (SEQ ID NO: 20): 5′-CAGCTCGAGCTGCTCGTTCTTCAGCACGCG-3 ′

  For cloning of the CaM and M13 genes, the same oligo DNA (SEQ ID NOs: 9-14) as in Example 1 is used.

(Procedure 2)
Each fragment of Renilla luciferase was cloned by PCR using the above oligo DNA. HRluc (manufactured by Promega Corp.) was used as a template. Using the oligo DNAs of SEQ ID NOs: 17 and 18 as primers, the N-terminal fragment gene for circular permutation sensor protein (cpRNLuc: including amino acids 1 to 91 of hRluc gene) was amplified, and SEQ ID NO: 19 and Twenty oligo DNAs were used to amplify a terminal fragment gene (cpRCLUc: containing amino acids 92 to 311 of the hRluc gene).

  In addition, CaM and M13 are cloned from the chameleon gene (YC2.1) in the same manner as in Example 1, so that the CaM-M13 gene, CaM-M13d10 gene, CaM-M13d05 gene, CaM-M13a05 gene and CaM-M13a10 gene can be obtained. The gene was amplified.

(Procedure 3)
Each gene amplified as described above was inserted into an expression plasmid to prepare a calcium sensor protein expression plasmid. Specifically, between the BamHI site and EcoRI site of pcDNA3.1 (manufactured by Invitrogen), which is a plasmid for animal cell expression, the cpRNLuc gene and the cpRCLUc gene are used as a luminescent enzyme fragment as a calcium binding region and an interaction region. CaM-M13 gene, CaM-M13d10 gene, CaM-M13d05 gene, CaM-M13a05 gene or CaM-M13a10 gene are inserted, and plasmid pcDNA / cphRL_C_CaM-M13_N, pcDNA / cphRL_C_CaM-M13d10_N, pcDNA / cphRLpC_05, cphRL_C_CaM-M13a05_N and pcDNA / cphRL_C_CaM-M13a10_N It was manufactured.

[Preparation 2]
In order to measure the function of the calcium sensor protein, an experimental system in which calcium ions are allowed to flow by stimulating cells with serotonin is used. Therefore, the gene of 5HT2A receptor, which is a serotonin receptor, was cloned in the same manner as in Example 1.

[Experiment]
Using the calcium sensor protein expression plasmid and the human 5HT2A receptor expression plasmid prepared as described above, luminescence imaging of calcium concentration fluctuations in HEK293 cells was performed.

(Procedure 1)
HEK293 cells obtained from ATCC were 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 2)
Each expression plasmid was introduced into the cells as follows. Cells were seeded in a 35 mm diameter glass bottom dish at a cell density of 2 × 10 ⁵ / dish and cultured overnight in a 5% CO ₂ incubator, and then a plasmid pcDNA / 5HT2AR for expressing human 5HT2A receptor and a plasmid for expressing calcium sensor protein pcDNA / cphRL_C_CaM-M13_N, pcDNA / cphRL_C_CaM-M13d10_N, pcDNA / cphRL_C_CaM-M13d05_N, the pcDNA / cphRL_C_CaM-M13a05_N and pcDNA / cphRL_C_CaM-M13a10_N, perform transfection using FuGENE HD (Roche), 5% CO ₂ Cultured overnight in incubator.

(Procedure 3)
The emission image was taken as follows. Coelenterazine 50 μM (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), and time-lapse photography of luminescence images was performed at intervals of 5 seconds. The personal computer configured as the image analysis apparatus 110 using the EM-CCD camera iXon (manufactured by Andor) with the magnification of the objective lens being 40 times, the exposure time being 5 to 10 seconds, the binning being 1 × 1, as the conditions for the light emission observation Incorporated.

(Procedure 4)
Two minutes after the start of time-lapse photography, stimulation was performed with serotonin 5HT (final concentration 10 μM), and then time-lapse photography of the luminescence image was performed. This stimulation causes calcium ions to flow into the cell.

(Procedure 5)
A plurality of ROIs (Region of Interest) are designated for each light emission image photographed in step 3 (FIG. 9), and a plurality of ROIs are designated for each light emission image photographed in step 4 ( FIG. 10). Furthermore, the emission intensity of each designated ROI was measured based on each emission image, and the change over time in the emission intensity was displayed in a graph (FIG. 11). 9 to 11 show the results when cphRL_C_CaM-M13_N is expressed in cells. The analysis of the luminescence image was performed using Metamorph software (made by Universal Imaging) that functions as the image analysis unit 112c.

[result]
As shown in FIGS. 9-11, when the calcium sensor protein cphRL_C_CaM-M13_N (SEQ ID NO: 22) using the circular permutation type Renilla luciferase gene is used, a change in luminescence intensity is detected before and after the serotonin 5HT stimulation. We were able to. That is, the luminescence intensity observed before stimulation (FIG. 9) increased after stimulation (FIG. 10). From the graph, it can be seen that the emission intensity greatly increases immediately after 5HT stimulation (horizontal axis 2-3 minutes and 17-18 minutes) (FIG. 11). Thus, it was found that by using cphRL_C_CaM-M13_N, the calcium response to stimulation can be detected at the single cell (single cell) level as an increase in luminescence intensity.

  On the other hand, when pcDNA / cphRL_C_CaM-M13d10_N, pcDNA / cphRL_C_CaM-M13d05_N, pcDNA / cphRL_C_CaM-M13a05_N and pcDNA / cphRL_C_CaM-M13a10_N were used, the luminescence image was weak and the luminescence image could not be obtained.

  Moreover, when the change of the emitted light intensity in each cell was seen, it turned out that there is big dispersion | variation in the response between cells in 5HT stimulation (FIG. 11). Previous studies on intracellular signal transduction mechanisms via calcium have revealed that the meaning of the signal differs depending on the intracellular distribution or time course of changes in calcium concentration. 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 case of the method using the calcium sensor protein according to the present invention, it was found that 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 experiments of Examples 1 and 2, accurate quantitative results from each cell can be obtained by the method using the calcium sensor protein according to the present invention without affecting the intracellular information transmission. As a result, it can be seen that changes in calcium concentration in individual cells can be observed over time with good reproducibility.

[Other Embodiments]
The present invention may be implemented in various different embodiments other than the above-described embodiments within the scope of the technical idea described in the claims.

  For example, in the above-described embodiment, the N-terminal fragment and the C-terminal fragment of the luminescent enzyme that are divided so that the luminescent enzyme activity is recovered by binding to each other, calmodulin that is a calcium-binding protein, and calcium-dependent protein. Although a fusion protein comprising a peptide (M13) that binds to calmodulin is described as a calcium sensor protein, the present invention is not limited to this, but is an N-terminal fragment and a C-terminal fragment of a luminescent enzyme, and a calcium-binding protein. Instead of a fusion protein comprising calmodulin and a peptide (M13) that binds to calmodulin in a calcium-dependent manner, calcium sensor proteins disclosed in Patent Document 2, Patent Document 3, Non-Patent Document 2, and the like may be used.

Moreover, according to the above-mentioned description, 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 amino acid sequence of an N-terminal fragment and a C-terminal fragment of the luminescent enzyme, calmodulin that is a calcium-binding protein, and a peptide (M13) that binds to calmodulin in a calcium-dependent manner, the N-terminal of the luminescent enzyme A polypeptide characterized in that the side fragment and the C-terminal fragment interact non-covalently, and the non-covalent interaction is adjustable.
[Polypeptide Aspect 2] (Function: Action of CaM-M13)
The polypeptide according to aspect 1, wherein the calcium-binding protein calmodulin and a peptide (M13) that binds to calcium in a calcium-dependent manner detectably change 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 calcium)
The polypeptide according to Aspect 2, wherein the exogenous factor is calcium.
[Polypeptide Aspect 5] (Function: Luminescence)
4. The polypeptide according to aspect 3, which emits light by the non-covalent interaction.

As described above in detail, the calcium analysis method according to the present invention can be suitably used in various fields such as biotechnology, pharmaceuticals, and medicine.
[Appendix] The invention described in the claims at the beginning of the application will be added below.
[Item 1] A calcium sensor protein comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, a calcium binding region, and an interaction region capable of reversibly binding to or dissociating from the calcium binding region, A cell production step for producing a cell containing a calcium sensor protein, wherein the calcium binding region and the interaction region are arranged between the C-terminal fragment and the N-terminal fragment;
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 calcium in the cell based on the luminescent image taken in the imaging step;
A method for analyzing intracellular calcium.
[Item 2] The intracellular calcium analysis method according to Item 1, wherein the calcium-binding region is derived from calmodulin.
[Item 3] The method for analyzing intracellular calcium according to Item 1 or 2, wherein the interaction region is an M13 peptide.
[Item 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
Item 4. The method for analyzing intracellular calcium according to any one of Items 1 to 3.
[Item 5] The luminescent enzyme is Renilla luciferase,
The N-terminal fragment comprises the amino acid sequence of amino acid numbers 1 to 91 of Renilla luciferase;
The C-terminal fragment contains the amino acid sequence of amino acid numbers 92 to 311 of Renilla luciferase
Item 4. The method for analyzing intracellular calcium according to any one of Items 1 to 3.
[Item 6] The intracellular calcium analysis method according to any one of Items 1 to 5, wherein the N-terminal fragment and the C-terminal fragment are circularly permuted with respect to the original arrangement of the fermentation enzyme.
[Item 7] The intracellular calcium analysis method according to any one of Items 1 to 6, wherein the imaging step includes repeatedly capturing the luminescent image.
[Item 8] The imaging step is simultaneously performed on a plurality of different cells,
The analysis step is performed for each cell;
Item 8. The intracellular calcium analysis method according to any one of Items 1 to 7.
[Item 9] The intracellular calcium analysis method according to any one of Items 1 to 8, wherein the calcium sensor protein has an amino acid sequence represented by SEQ ID NO: 21 or 22.
[Item 10] A calcium sensor protein comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, a calcium binding region, and an interaction region capable of reversibly binding to or dissociating from the calcium binding region, The calcium binding region and the interaction region are disposed between the C-terminal fragment and the N-terminal fragment, and the N-terminal fragment and the C-terminal fragment are circularly permuted with respect to the original position in the fermentation enzyme. Calcium sensor protein.
[Item 11] The calcium sensor protein according to Item 10, wherein the calcium-binding region is derived from calmodulin.
[Item 12] The calcium sensor protein according to Item 10 or 11, wherein the interaction region is an M13 peptide.
[Item 13] 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
Item 13. The calcium sensor protein according to any one of Items 10 to 12.
[Item 14] The luminescent enzyme is Renilla luciferase,
The N-terminal fragment comprises the amino acid sequence of amino acid numbers 1 to 91 of Renilla luciferase;
The C-terminal fragment contains the amino acid sequence of amino acid numbers 92 to 311 of Renilla luciferase
Item 13. The calcium sensor protein according to any one of Items 10 to 12.
CLAIM | ITEM 15 Calcium sensor protein of any one of claim | item 10 thru | or 14 which has an amino acid sequence shown by sequence number 21 or 22.

100 Luminescence observation system
102 cells
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 calcium sensor protein comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, a calcium binding region, and an interaction region capable of reversibly binding or dissociating with the calcium binding region, wherein the calcium binding region and A cell production step of producing a cell containing a calcium sensor protein disposed between the C-terminal fragment and the N-terminal fragment of the interaction region;
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 calcium in the cell based on the luminescent image imaged in the imaging step,
The calcium sensor protein has the amino acid sequence shown in SEQ ID NO: 21 or 22 .
Intracellular calcium analysis method. The intracellular calcium 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 intracellular calcium analysis method according to claim 1 or 2 , wherein the analysis step is performed for each cell. A calcium sensor protein comprising an N-terminal fragment of a luminescent enzyme, a C-terminal fragment of the luminescent enzyme, a calcium binding region, and an interaction region capable of reversibly binding or dissociating with the calcium binding region, wherein the calcium binding region and said interaction region is disposed between the N-terminal fragment and the C-terminal fragment, the N-terminal fragment and the C-terminal fragment is circularly permutated substituted for the original arrangement in the light - emitting enzyme A calcium sensor protein,
The luminescent enzyme is a luminescent enzyme selected from firefly luciferase and Renilla luciferase,
The calcium binding region is derived from calmodulin;
The interaction region is M13 peptide ;
A calcium sensor protein having the amino acid sequence shown in SEQ ID NO: 21 or 22 .

Images