JP2010213661A - Method for analyzing amount of receptor expression, and luminous protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing the amount of receptor expression, etc., capable of obtaining an accurate quantitative result for each of cells regarding the expression amount of receptors without being affected by the promotor activity of the cells, and as a result, capable of observing the changes of expressed amounts and distribution of the receptors in good reproducibility in each of the cells. <P>SOLUTION: The method for analyzing the amount of receptor expression includes preparing the cells containing luciferase, adding luciferin to the prepared cells, adding dopamine to the cells after adding the luciferin, measuring the light-emitting amounts of the cells after adding the dopamine, analyzing the concentrations of ATP in the cells based on the measured light-emitting amounts, and analyzing the expressed amounts of D2 receptor based on the analyzed concentration of the ATP. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a receptor expression level analysis method for analyzing the expression level of a receptor (receptor gene) such as a D2 receptor by noninvasively analyzing the concentration of intracellular ATP and expression of the receptor. It relates to a photoprotein for analyzing the amount.

  Many hormones and neurotransmitters act on G protein-coupled receptors and cause various cellular activities through intracellular second messengers. Neurotransmitters that act on G protein-coupled receptors are involved in the regulation of brain functions over a wide range of human emotions, moods, dependence, cognition, learning, memory, etc. Among them, monoamines and their receptors are It is targeted for treatment of mental disorders such as manic depression, schizophrenia, mood disorders, autism, attention deficit / hyperactivity disorder. Such higher-order functions of the brain are controlled by the temporal and spatial concentration distributions of transmitter substances and their receptors in the brain. Therefore, it is necessary to capture in real time the temporal and spatial changes of G protein-coupled receptors in the brain when analyzing information transmission mechanisms related to specific emotional activities.

  Conventionally, the amount and distribution of G protein-coupled receptors have been measured using radiolabeled compounds. This is because the ligand for the receptor is radiolabeled, and the labeled ligand and test chemical are bound competitively to the receptor present in cells, tissues, individuals or their crude fractions, and then bound. And a method of knowing the binding between the test chemical and the receptor by measuring the amount of the released ligand. Furthermore, in recent years, new receptor detection methods have been found. For example, Bertran-Gonzalez et al. Produced a transgenic mouse in which a fluorescent protein EGFP (Enhanced Green Fluorescent Protein) gene was linked downstream of the promoter region of the D1 and D2 receptor genes, which are subtypes of the dopamine receptor. Thus, fluorescence observation of the cells or tissues of the transgenic mouse is disclosed (see Non-Patent Document 1). In this transgenic mouse, the fluorescent protein is expressed in the cell in which the promoter of the D1 or D2 receptor gene works, that is, the cell in which the D1 or D2 receptor is expressed. It is revealed that cells expressing D1 or D2 receptor can be observed.

Bertran-Gonzalez J, Bosch C, Maroteaux M, Materials M, Herve D, Valent E, Girt JA. , “Oposing patterns of signaling activation in dopamin D1 and D2 receptor-expressing strategic neurons in response to cocaine and haloperol. 28, no. 22, pp5671-5585, 2008

However, in the conventional receptor expression / distribution analysis method using a radiolabeled ligand, it cannot be determined whether the test chemical substance is an agonist or an antagonist, and the resolution at the time of detection of the labeled ligand is poor. There was a problem that the expression level and distribution / functional state (response) of the body could not be accurately shown.
Moreover, in the conventional receptor expression / distribution analysis method using a probe in which a fluorescent protein is linked to the promoter of the receptor gene, the promoter activity and the gene expression level do not necessarily match. There was a problem that the functional state (response) could not be accurately shown.

  The present invention has been made in view of the above problems, and can obtain an accurate quantitative result from each cell regarding the expression level of the receptor without being influenced by the promoter activity of the cell. It is an object of the present invention to provide a receptor expression level analysis method capable of observing changes in the expression level and distribution of receptors in cells with good reproducibility, and a photoprotein for analyzing the expression level of receptors.

  In order to solve the above-described problems and achieve the object, a receptor expression level analysis method according to the present invention is a receptor expression level analysis method for analyzing the expression level of a receptor in a cell, which is ATP-dependent. A production process for producing the cells containing a reporter protein, a luminescence treatment process for performing a predetermined treatment for causing the cells produced in the production process to emit light from outside the cell, and after the luminescence treatment process A substance addition step of adding a predetermined receptor binding substance from the outside of the cell to the cell, a measurement step of measuring the amount of luminescence of the cell after the substance addition step is performed, and the measurement step A concentration analysis step for analyzing the concentration of the ATP in the cell based on the amount of luminescence, and an expression for analyzing the expression level of the receptor based on the concentration of the ATP analyzed in the concentration analysis step Characterized in that it comprises an analysis step. Here, in the present invention, the reporter protein includes a fluorescent protein and a photoprotein. The photoprotein is specifically a bioluminescent protein. Moreover, the predetermined treatment for causing light emission includes, for example, adding a luminescent substrate or irradiating excitation light.

  The receptor expression level analysis method according to the present invention is characterized in that, in the receptor expression level analysis method described above, in the preparation step, the reporter protein is the ATP-dependent photoprotein. .

  The receptor expression level analysis method according to the present invention is characterized in that, in the receptor expression level analysis method described above, the photoprotein emits light by interacting with the ATP in the cell. And

  The receptor expression level analysis method according to the present invention is characterized in that, in the receptor expression level analysis method described above, the photoprotein is a firefly luciferase or a lightning luciferase.

  The receptor expression level analysis method according to the present invention is characterized in that, in the receptor expression level analysis method described above, in the preparation step, the cell contains the receptor.

  The receptor expression level analysis method according to the present invention is characterized in that, in the receptor expression level analysis method described above, in the preparation step, the cell is a gene into which the receptor gene has been introduced. And

  The receptor expression level analysis method according to the present invention is the receptor expression level analysis method described above, wherein the measurement step further includes a photon amount measurement step of measuring the photon amount of the ATP-dependent luminescent label. Including.

  The receptor expression level analysis method according to the present invention is characterized in that, in the receptor expression level analysis method described above, the measurement step further includes an imaging step of imaging the ATP-dependent luminescent label. And

  The receptor expression level analysis method according to the present invention is the receptor expression level analysis method described above, wherein the imaging step is performed simultaneously on a plurality of different cells, and the concentration analysis step includes It further includes a collation step of collating the plurality of luminescent images captured in the imaging step for each of the cells.

  The receptor expression level analysis method according to the present invention is characterized in that, in the receptor expression level analysis method described above, the measurement step is repeatedly executed.

  The receptor expression level analysis method according to the present invention is characterized in that, in the receptor expression level analysis method described above, the receptor is a D2 receptor.

  The receptor expression level analysis method according to the present invention is characterized in that, in the receptor expression level analysis method described above, in the substance addition step, the receptor binding substance is dopamine.

  In addition, the present invention relates to a photoprotein, and the photoprotein according to the present invention is an ATP-dependent photoprotein for analyzing the expression level of a receptor.

  Moreover, the photoprotein according to the present invention is characterized in that, in the photoprotein described above, the photoprotein emits light by interacting with ATP.

  The photoprotein according to the present invention is characterized in that, in the photoprotein described above, the receptor is a D2 receptor.

  According to the present invention, a cell containing an ATP-dependent reporter protein is prepared, the prepared cell is subjected to a predetermined treatment for causing light emission from outside the cell, and the cell after the treatment is applied to the cell. A predetermined receptor-binding substance is added from the outside, the amount of luminescence of the cell after the addition is measured, the concentration of ATP in the cell is analyzed based on the measured amount of luminescence, and the analyzed ATP concentration Based on the analysis of the receptor expression level, it is possible to obtain an accurate quantitative result from each cell regarding the expression level of the receptor without being influenced by the promoter activity of the cell. The effect is that the change in the expression level and distribution of the receptor can be observed with good reproducibility.

  According to this invention, since the reporter protein is an ATP-dependent photoprotein, an accurate quantitative result from each cell regarding the expression level of the receptor can be obtained using the ATP detection principle by bioluminescence, As a result, it is possible to observe changes in receptor expression level and distribution in individual cells with good reproducibility.

  According to this invention, since the photoprotein emits light by interacting with intracellular ATP, the ATP concentration can be detected by luminescence with enhanced interaction in the presence of ATP. .

  According to the present invention, since the photoprotein is a firefly luciferase or a light click luciferase, the ATP concentration can be calculated by measuring the amount of luminescence.

  According to the present invention, since the cell contains a receptor, there is an effect that the experiment can be facilitated by using an easy-to-handle cell that artificially includes the receptor to be analyzed.

  According to the present invention, since the cell is introduced with a receptor gene, the experiment can be facilitated using an easy-to-handle cell artificially including the receptor to be analyzed. It is possible to obtain a response close to.

  According to the present invention, since the photon amount of the ATP-dependent luminescent label is measured, the luminescence amount from the entire cell or the entire cell population can be obtained using the photon counting technique.

  According to this invention, since an ATP-dependent luminescent label is imaged, there is an effect that it is possible to obtain the amount of luminescence from individual cells using a luminescence microscope.

  According to this invention, since imaging is performed simultaneously on a plurality of different cells and the plurality of captured luminescent images are collated for each cell, the amount of luminescence from each cell can be obtained individually and simultaneously. There is an effect that can be done.

  According to the present invention, since the measurement of the light emission amount is repeatedly executed, it is possible to observe the temporal change of the light emission amount.

  According to the present invention, since the receptor is a D2 receptor, an accurate quantitative result from each cell regarding the expression level of the D2 receptor can be obtained, and as a result, the expression of the D2 receptor in each individual cell can be obtained. There is an effect that changes in the amount and distribution can be observed with good reproducibility.

  According to this invention, since the receptor-binding substance is dopamine, there is an effect that the activity of the D2 receptor can be observed.

  According to the present invention, since the photoprotein is ATP-dependent and is used for analyzing the expression level of the receptor, the photoprotein is used without being influenced by the promoter activity of the cell. An accurate quantitative result from each cell regarding the expression level can be obtained, and as a result, changes in receptor expression level and distribution in individual cells can be observed with good reproducibility.

  According to the present invention, the photoprotein emits light by interacting with ATP. Therefore, when the photoprotein is used, the ATP concentration can be detected by luminescence with enhanced interaction in the presence of ATP. There is an effect.

  According to the present invention, the photoprotein is used for analyzing the expression level of the D2 receptor. Therefore, when the photoprotein is used, an accurate quantitative result from each cell regarding the expression level of the D2 receptor is obtained. As a result, it is possible to observe changes in the expression level and distribution of the D2 receptor in individual cells with good reproducibility.

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. FIG. 4 is a block diagram illustrating an example of the configuration of the image analysis device 110 of the light emission observation system 100. FIG. 5 is a diagram showing the principle of ATP detection using the luciferin-luciferase reaction. FIG. 6 is a graph showing a time-dependent change in luminescence intensity of luciferase by dopamine stimulation in HEK293 cells introduced with firefly luciferase and D1 receptor or D2 receptor, as measured by a luminometer. FIG. 7 is a diagram showing a luminescence image before stimulation with dopamine containing HEK293 cells into which firefly luciferase and D2 receptor have been introduced. FIG. 8 is a diagram showing a luminescence image 10 minutes after stimulation with dopamine containing HEK293 cells into which firefly luciferase and D2 receptor have been introduced. FIG. 9 is a graph showing a change with time of the luminescence intensity of luciferase by dopamine stimulation in selected cells, measured by a luminometer.

  Embodiments of a receptor expression level analysis method and a photoprotein for analyzing receptor expression levels according to the present invention will be described below in detail with reference to the drawings. In addition, this invention is not limited by this Embodiment. Here, in this embodiment, a case where the expression level of the D2 receptor is analyzed using a photoprotein (specifically, a bioluminescent protein) will be described as an example. However, the present invention also applies to fluorescent proteins and other receptors. It can be implemented similarly.

[1. Constitution]
First, the structure of the luminescence observation system 100 used in the receptor expression level analysis method according to the present invention will be described with reference to FIG. 1, FIG. 2, and FIG. FIG. 1 is a diagram illustrating an example of the overall configuration of the light emission observation system 100.

  As shown in FIG. 1, a luminescence observation system 100 includes a container 103 (specifically, a petri dish, a slide glass, a microplate, a gel support, a fine particle carrier, etc.) that contains cells 102, and a stage 104 on which the container 103 is arranged. And a light emission image capturing unit 106 for measuring weak light emission, and an image analysis device 110. Note that the luminescent image capturing unit 106 may be disposed below the stage 104. Thereby, disturbance light from above the sample due to opening and closing of a cover (not shown) can be completely blocked, and as a result, 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.

  The cell 102 is, for example, a living cell that is luminescence-labeled so as to emit light depending on an ATP (adenosine triphosphate) concentration. Here, as a protein that emits light depending on the ATP concentration (photoprotein), in addition to firefly luciferase, for example, lightning luciferase or a modified form thereof may be used. Here, the photoprotein is expressed in the cell 102 by introducing an expression vector containing the polynucleotide encoding the photoprotein, which is configured to express the photoprotein, into the cell 102. May be. The cell 102 is given a predetermined luminescent substrate (for example, luciferin) and a predetermined stimulus (for example, drug stimulus) from outside the cell 102.

The luminescence image capturing unit 106 is specifically an upright luminescence microscope and captures a luminescence 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 (charge-coupled device) camera 106c, and an imaging lens 106f. Specifically, it is preferable that 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. 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, and specifically, a memory device such as a random access memory (RAM) or a read-only memory (ROM), a fixed disk device such as a hard disk, a flexible disk, an optical disk, or the like is used. Can do. 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 a control program such as an OS (Operating System), a program that defines various processing procedures, and necessary data, and executes various processes based on these programs. . 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 luminescent image capturing instruction unit 112a instructs the CCD camera 106c to capture a luminescent 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. The control unit 112 controls the emission image capturing instruction unit 112a to cause the CCD camera 106c to repeatedly capture the emission image and the bright field image of the cell 102, and controls the emission image acquisition unit 112b to control the captured emission image and the bright image. A field-of-view image is acquired every time it is imaged or after all imaging is completed.

  The image analysis unit 112c measures the luminescence intensity of the luminescence emitted from the cells 102 over time based on the plurality of luminescence images acquired by the luminescence image acquisition unit 112b. The analysis result output unit 112d outputs the analysis result from the image analysis unit 112c to the output device 124. Specifically, the analysis result output unit 112d graphs and displays on the output device 124 time-series data regarding the emission intensity of the luminescence emitted from the cell 102 obtained by the image analysis unit 112c.

[2. Example]
Examples of the present embodiment will be described below with reference to FIGS. Here, FIG. 5 is a diagram showing the principle of ATP detection using the luciferin-luciferase reaction.

  In the luciferin-luciferase reaction, luciferin and ATP react to form adenylate luciferin, and when the adenylate luciferin and oxygen are degraded by oxidative decarboxylation in the presence of luciferase, oxyluciferin, AMP (adenosine monophosphate) and carbon dioxide Carbon is produced. Part of the energy obtained in the course of this reaction emits light. By quantifying this luminescence, ATP can be quantified.

  In this example, an analysis method for analyzing the expression level of the D2 receptor using the above-described ATP detection principle will be described. The flow of the experiment (procedure) in this example is as follows.

[Preparation: Cloning of human D1 receptor gene and D2 receptor gene]
[Procedure 1]
In order to prepare the human D1 receptor gene and the D2 receptor gene, a synthetic oligo DNA (deoxyribonucleic acid) used for PCR (polymerase chain reaction) was prepared with the sequences shown below.
[Synthetic oligo DNA sequence for preparing human D1 receptor gene]
HU_D1R_Fw (SEQ ID NO: 1): 5′-GCCACATGAGGACTCTGAACACCCTCTCC-3 ′
HU_D1R_Rv (SEQ ID NO: 2): 5′-TCAGGTTGGGTGCTGACCGTTTTGTGTGAT-3 ′
[Synthetic oligo DNA sequence for preparing human D2 receptor gene]
HU_D2R_Fw (SEQ ID NO: 3): 5′-GCCACATGGATCCACTGAATCTGCTCGG-3 ′
HU_D2R_Rv (SEQ ID NO: 4): 5′-TCAGCAGTGGAGGATCTTCAGGAAGGCCT-3 ′

[Procedure 2]
Using a human brain cDNA library (manufactured by Takara Bio Inc.) as a template, the D1 receptor gene (including the region corresponding to the full length of the human D1 receptor gene) and the D2 receptor gene (the full length of the human D2 receptor gene) Corresponding region was amplified by PCR using the synthetic oligo DNA as a primer.

[Procedure 3]
After subcloning the D1 receptor gene and D2 receptor gene amplified by PCR into the pBluescript II vector, the sequence of the prepared DNA was confirmed by DNA sequencing.

[Procedure 4]
The respective genes were incorporated into mammalian cell expression vector pcDNA3.1 (+) (manufactured by Invitrogen) to prepare plasmid DcDNA / D1R for human D1 receptor expression and plasmid pcDNA / D2R for expression of human D2 receptor.

[Experiment 1: Measurement of luminescence level by dopamine stimulation in HEK293 cells expressing D2 receptor]
[Procedure 1: Culture of HEK293 cells]
HEK293 cells obtained from ATCC (American Type Culture Collection) were cultured in Earle's MEM / Medium (GI) supplemented with 10% Fetal Bovine Serum and 1 × Nonesential amino acids in a 5% CO2 incubator. .

[Procedure 2: Introduction of luciferase expression plasmid]
HEK293 cells were seeded at a cell density of 2 × 10 ⁵ / dish in a 35 mm diameter glass bottom dish, cultured overnight in a 5% CO 2 incubator, and the plasmid pGL3-control (Promega) for luciferase expression and human D1 reception The plasmid pcDNA / D1R for body expression or the plasmid pcDNA / D2R for expression of the human D2 receptor was mixed, transfected using FuGENE HD (Roche), and cultured overnight in a 5% CO2 incubator. The amino acid sequence of human D1 receptor is specifically the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of human D2 receptor is specifically the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of luciferase is specifically the amino acid sequence of SEQ ID NO: 7.

[Procedure 3: Measurement of luminescence]
After adding 2 mM luciferin (Promega) in the medium and allowing to stand for 1 hour, the culture dish is set in a culture dish-compatible luminometer Kronos (Ato) and the amount of luminescence is measured at intervals of 3 minutes. It was. As conditions for measuring luminescence, the temperature in the apparatus was 36 ° C., and the exposure time of each culture dish was 10 seconds.

[Procedure 4: Measurement of light emission by dopamine stimulation]
One hour after the start of measurement of luminescence, stimulation was performed with dopamine (DA: final concentration 10 μM), and the luminescence was measured over time.

[Procedure 5: Graphical display of change in light emission over time]
The time-dependent change of the measured luminescence amount (luminescence intensity) was displayed as a graph (see FIG. 6). In FIG. 6, the graph corresponding to D1R shows the change over time of the emission intensity in the D1 receptor, and the graph corresponding to D2R shows the change over time in the emission intensity of the D2 receptor.

[Result of Experiment 1]
As a result of the above experiment, as shown in FIG. 6, it was possible to detect a change in the response of the cells to the dopamine stimulation on the D2 receptor. On the other hand, when the change of the luminescence intensity with respect to dopamine stimulation to the D1 receptor shown in FIG. 6 was observed, no cellular response to dopamine stimulation was observed. Thus, by analyzing the change in the amount of luminescence using a luminometer, the response of the entire cell population could be observed in real time, and the expression level of the D2 receptor could be studied in detail.

[Experiment 2: Dopamine-stimulated luminescence imaging in HEK293 cells expressing D2 receptor]
[Procedure 1: Culture of HEK293 cells]
HEK293 cells obtained from ATCC (American Type Culture Collection) were cultured in Earle's MEM / Medium (GI) supplemented with 10% Fetal Bovine Serum and 1 × Nonesential amino acids in a 5% CO2 incubator. .

[Procedure 2: Introduction of luciferase and dopamine receptor expression plasmid]
HEK293 cells were seeded at a cell density of 2 × 10 ⁵ / dish in a 35 mm diameter glass bottom dish, cultured overnight in a 5% CO 2 incubator, and the plasmid pGL3-control (Promega) for luciferase expression and human D2 reception The somatic expression plasmid pcDNA / D2R was mixed, transfected using FuGENE HD (Roche), and cultured overnight in a 5% CO2 incubator. The amino acid sequence of the human D2 receptor is specifically the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of luciferase is specifically the amino acid sequence of SEQ ID NO: 7.

[Procedure 3: Shooting a luminescent image]
After adding 2 mM luciferin (Promega) in the medium and allowing to stand for 1 hour, the culture dish was set on a luminescence microscope “LV (LUMINOVIEW) -200” (Olympus), and the luminescence images were displayed at 20-second intervals. Time-lapse photography was performed. As the light emission observation conditions, the temperature in the apparatus was 36 ° C., the magnification of the objective lens was 60 times, the exposure time was 10 seconds, and the binning was 1 × 1. An EM-CCD camera iXon (manufactured by Andor Co.) was used as the CCD camera 106c, and the luminescence image was taken into a personal computer configured as the image analysis device 110.

[Procedure 4: Shooting Luminescent Image by Dopamine Stimulation]
Five minutes after the start of time-lapse photography, stimulation was performed with dopamine (DA: final concentration 10 μM), and then time-lapse photography of the luminescence image was performed.

[Procedure 5: Graphical display of change in light emission over time]
As shown in the figure, a plurality of ROIs (Region of Interest) are designated for each luminescence image (see FIG. 7) photographed before dopamine stimulation, and each luminescence image (see FIG. 8) photographed after dopamine stimulation. A plurality of ROIs are designated as shown in FIG. The emission intensity of each designated ROI was measured based on each emission image, and the change over time of the measured emission intensity was displayed in a graph (see FIG. 9). The analysis of the luminescence image was performed using Metamorph software (made by Universal Imaging) that functions as the image analysis unit 112c. In FIG. 9, each graph corresponding to each of Cell 1 to Cell 5 shows a change over time in the emission intensity of each ROI corresponding to each of numbers 1 to 5 in FIGS. 7 and 8.

[Result of Experiment 2]
As a result of the above experiment, as shown in FIG. 7, FIG. 8, and FIG. 9, the change in the response of the cells to the dopamine stimulation to the D2 receptor could be detected at the single cell level (one cell level). Moreover, when the change of the emitted light intensity in each cell shown by FIG. 9 was seen, it turned out that there is big dispersion | variation in the response between cells with respect to dopamine stimulation. Previous studies of intracellular signal transduction mechanisms via dopamine receptors have revealed that the meaning of the signal differs depending on the number or distribution of stimulated dopamine receptors. In the conventional analysis of changes in the amount of luminescence using a luminometer, the response of the entire cell population can be observed, but the response of individual cells cannot be analyzed. However, with the luminescence microscope “LV-200”, changes in the living body can be observed in real time for each cell, and the function of signals in tissues or cell populations can be studied in detail.

[Summary of this example]
As described above, according to this example, it is possible to obtain an accurate quantitative result from a cell population or each cell without being influenced by the receptor gene promoter activity of the cell. As a result, D2 in the cell population or individual cells can be obtained. The expression level and distribution of the receptor could be observed over time with good reproducibility.

[3. Other Embodiments]
In the above-described embodiment, firefly luciferase (SEQ ID NO: 7) has been mainly described as an ATP sensor protein. However, the present invention is not limited to this, and instead of firefly luciferase, a light click luciferase (sequence) No. 8) and variants thereof may be used.

  In the above-described embodiment, the description has been made mainly using the established cell or cell population, but the present invention is not limited to this, and the cell and cell population derived from an individual, a section, a tissue, Individuals may be used.

  As described above, the receptor expression level analysis method and photoprotein 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 (15)

A receptor expression level analysis method for analyzing a receptor expression level in a cell,
A production step of producing said cells comprising an ATP-dependent reporter protein;
A luminescence treatment step of performing a predetermined treatment for causing the cells produced in the production step to emit light from outside the cells;
A substance addition step of adding a predetermined receptor binding substance from the outside of the cell to the cell after the luminescence treatment step is performed;
A measurement step of measuring the amount of luminescence of the cells after the substance addition step is performed;
A concentration analysis step of analyzing the concentration of the ATP in the cell based on the amount of luminescence measured in the measurement step;
An expression level analysis step of analyzing the expression level of the receptor based on the concentration of the ATP analyzed in the concentration analysis step;
A receptor expression level analysis method comprising the steps of: In the production step, the reporter protein is the ATP-dependent photoprotein,
The receptor expression level analysis method according to claim 1. The photoprotein interacts with the ATP in the cell and emits light;
The receptor expression level analysis method according to claim 2. The photoprotein is a firefly luciferase or a lightning luciferase;
The receptor expression level analysis method according to claim 2 or 3. In the production step, the cell contains the receptor.
The receptor expression level analysis method according to any one of claims 1 to 4, wherein: In the production step, the cell is a cell into which the receptor gene has been introduced,
The receptor expression level analysis method according to any one of claims 1 to 4, wherein: The measurement step further includes a photon amount measurement step of measuring a photon amount of the ATP-dependent luminescent label,
The receptor expression level analysis method according to any one of claims 1 to 6, wherein: The measurement step further includes an imaging step of imaging the ATP-dependent luminescent label;
The receptor expression level analysis method according to any one of claims 1 to 6, wherein: The imaging step is performed simultaneously on a plurality of different cells,
The concentration analysis step further includes a collation step of collating the plurality of luminescent images captured in the imaging step for each of the cells,
The receptor expression level analysis method according to claim 8, wherein: The measurement step is repeatedly performed;
The receptor expression level analysis method according to any one of claims 1 to 9. The receptor is a D2 receptor;
The receptor expression level analysis method according to any one of claims 1 to 10, wherein: In the substance addition step, the receptor binding substance is dopamine;
The receptor expression level analysis method according to claim 11. An ATP-dependent photoprotein,
For analyzing the expression level of the receptor,
A photoprotein characterized by Interact with ATP to emit light,
The photoprotein according to claim 13. The receptor is a D2 receptor;
The photoprotein according to claim 13 or 14, wherein