JP2016002071A - Method for analyzing biological sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method which enables analysis of a state change of a biological sample for a long period of time by using chemiluminescence.SOLUTION: An analysis method according to an embodiment includes the steps of: imaging a biological sample comprising a portion containing luciferase, luciferin, and luciferin-regenerating enzyme with time and acquiring a plurality of luminance images; and associating the brightness of a region corresponding to the portion of each of the plurality of luminance images with the state of the portion or the state of the biological sample. Luciferin-regenerating enzyme protein according to an embodiment has an amino acid sequence described in SEQ ID NO:1. Nucleic acid according to an embodiment contains a luciferin-regenerating enzyme gene having a base sequence described in SEQ ID NO:2.

Description

  The present invention relates to a biological sample analysis method.

  Chemiluminescence of luciferin generated in the presence of luciferase is used to analyze in vivo activities such as intracellular signal transduction and gene expression. This analysis using chemiluminescence has advantages such as no cell damage caused by excitation light, no self-luminescence problem, and excellent quantitativeness.

  In general, in the above-described analysis using chemiluminescence, first, a cell into which a luciferase gene has been introduced or a cell into which a fusion protein of a luciferase and a protein to be analyzed has been prepared is prepared, and this cell is lysed to lyse the cell Then, luciferin, adenosine triphosphate (ATP) and the like are added to the cell lysate, and the amount of luminescence is measured with a luminometer using a photomultiplier tube. That is, the measurement of luminescence is performed after cell lysis. For this reason, the amount of luminescence at a certain point in time is obtained as an average value of all cells used for the measurement.

  An enzyme that converts oxyluciferin to luciferin has been found (Non-patent Document 1). Oxyluciferin is a substance that is generated as luciferin undergoes chemiluminescence, that is, as the oxidation of luciferin proceeds. This enzyme is named luciferin regenerating enzyme (LRE). It has been found that when this LRE is added to a solution that generates a chemiluminescent reaction catalyzed by luciferase, the amount of luminescence increases (FIG. 8 of Non-Patent Document 1).

  Utilizing the advantage of increasing the amount of luminescence of LRE, a method for measuring in vivo substances using LRE has been studied. Patent Document 1 discloses a method for measuring an ATP amount using LRE. In this method, a luminometer, a scintillation counter, a photometer and a photoemulsion film are shown as means for measuring luciferin-induced luminescence induced by luciferase (paragraph 0023). Patent Document 2 discloses a method for measuring intracellular ATP and / or gene expression using LRE. In this method, a luminometer is used as means for measuring luciferin luminescence (paragraphs 0094 and 0095). Therefore, in any case, the amount of luminescence is obtained as an average value at a certain point in time for all cells used in the measurement.

Japanese Patent No. 3983023 JP-T-2005-509426

J. Biol. Chem., September 2001, Vol.276, p.36508-36513

  The problem to be solved by the present invention is to provide a method capable of analyzing a state change of a biological sample over a long period of time by using chemiluminescence.

  The analysis method according to the embodiment includes imaging a biological sample including a portion including luciferase, luciferin, and luciferin regenerating enzyme over time to obtain a plurality of luminescence images, and each of the plurality of luminescence images. Associating the brightness of the region corresponding to the portion of the portion with the state of the portion or the state of the biological sample.

  The luciferin regenerating enzyme protein according to the embodiment has the amino acid sequence set forth in SEQ ID NO: 1.

  The nucleic acid according to the embodiment includes a luciferin regenerating enzyme gene having the base sequence set forth in SEQ ID NO: 2.

  According to the present invention, it is possible to analyze a change in the state of an individual biological sample over a long period of time.

FIG. 1 is a graph showing the influence of the luciferin regenerating enzyme according to the embodiment on the change over time of the luminescence amount of luciferase in vitro. FIG. 2 is a graph showing the influence of the luciferin regenerating enzyme according to the embodiment on the integrated value of the luciferase luminescence amount in vitro. FIG. 3 is a graph showing the prediction of the effect of the luciferin regenerating enzyme according to the embodiment on the integrated value of the luciferase luminescence amount in vivo. FIG. 4 is a graph showing a prediction of the effect of the luciferin regenerating enzyme according to the embodiment on the change over time of the luminescence amount of luciferase in vivo. FIG. 5 is a graph showing a prediction of comparison between the luciferin regenerating enzyme according to the embodiment and the conventional luciferin regenerating enzyme with respect to the influence of the luminescence amount of luciferase on the integrated value. FIG. 6 is a graph showing a prediction of comparison between the luciferin-regenerating enzyme according to the embodiment and the conventional luciferin-regenerating enzyme with respect to the influence of the luminescence amount of luciferase on the temporal change. FIG. 7 a is a graph showing the prediction of the measurement result of the fluctuation in the expression of the clock gene using the luciferin regenerating enzyme according to the embodiment. FIG. 7 b is a graph showing the prediction of the measurement result of the variation in the expression of the clock gene using the luciferin regenerating enzyme according to the embodiment. FIG. 8 is an image showing a prediction obtained by imaging cells that emit light in the method according to the embodiment. FIG. 9A is an image showing a prediction obtained by imaging cells that emit light in the method according to the embodiment. FIG. 9B is a graph showing a prediction that the amount of luminescence for each cell is measured over time by the method according to the embodiment.

  The first embodiment of the present invention is an analysis method.

  The analysis method according to the embodiment includes imaging a biological sample including a portion including luciferase, luciferin, and luciferin regenerating enzyme over time to obtain a plurality of luminescent images, and each of the luminescent images described above. Associating the brightness of the region corresponding to the part with the state of the part or the state of the biological sample. The “luminescence image” is an image including a distribution of brightness generated by chemiluminescence of luciferin.

  In this analysis method, for example, a first step of producing a biological sample containing luciferase and luciferin-regenerating enzyme, a second step of taking a luminescent image of the biological sample over time, and a plurality of luminescent images obtained by imaging Based on this, the third step of examining the change with time of the light emission amount of the biological sample is sequentially performed.

  In the first step, a biological sample containing luciferase and luciferin regenerating enzyme is prepared. The biological sample is, for example, a cell, an embryo, a tissue, or an individual. The cell is, for example, a cultured cell or a cell of a unicellular organism. Embryos include various cells or cell populations that are in the early stages of development of multicellular organisms. The tissue may be a tissue section. Individuals include individuals of unicellular organisms and individuals of multicellular organisms. When the biological sample is a cell, luciferase and luciferin regenerating enzyme are contained in the cell. When the biological sample is an embryo, tissue or individual, luciferase and luciferin regenerating enzyme are contained in the cells constituting these biological samples. The biological sample may contain two or more luciferases. For example, the biological sample may contain two or more luciferases that catalyze a distinguishable luminescent reaction. That is, two or more kinds of such luciferases can catalyze luminescent reactions with different luminescent colors, and can measure their active states.

  Luciferase generally refers to an enzyme that catalyzes a chemical reaction in which luminescence occurs. A substance that is a substrate for the enzyme is called luciferin. Luminescence occurs when luciferin undergoes a chemical change by the catalytic action of luciferase in the presence of ATP. Luciferase derived from various organisms can be used. For example, those derived from the firefly family belonging to the order of Coleoptera, those derived from the Iriomote family, and those derived from the rice family can be used.

  Luciferin regenerating enzyme (LRE) is an enzyme that catalyzes a reaction in which luciferin is generated from oxyluciferin as a reactant. LRE derived from various organisms can be used. For example, LRE derived from Genji fireflies or LRE derived from Heike fireflies can be used. Furthermore, LRE having the amino acid sequence shown in SEQ ID NO: 1 newly discovered by the present inventors may be used. That is, usable LREs are not limited to known ones.

  Luciferase and luciferin regenerating enzyme can be introduced into a biological sample by any method. For example, a gene can be introduced into a biological sample by using a general genetic engineering technique, and luciferase and luciferin regenerating enzyme can be expressed from the gene. Alternatively, these proteins may be directly introduced into a biological sample by, for example, a microinjection method. The luciferase and the luciferin regenerating enzyme are preferably positioned close to each other so that the luciferin oxidized by luciferase catalysis, ie, oxyluciferin, is quickly returned to luciferin by the luciferin regenerating enzyme.

  In the second step, a luminescent image of the biological sample is taken over time. As an apparatus used for imaging, an apparatus capable of acquiring a luminescent image is used. In particular, a microscope can be used. At the time of imaging, the biological sample is placed in an environment where a luminescence reaction can occur. For example, an appropriate amount of luciferin is added to a culture medium suitable for the biological sample to be used. Imaging can be performed over time at an arbitrary timing. For example, after giving a specific stimulus to a biological sample, imaging can be performed a plurality of times at regular time intervals. When imaging using a microscope, only a single cell or a plurality of cells may be placed in the field of view. It is preferable to acquire an image including a plurality of cells in that the overall state of the cells in the biological sample can be confirmed.

  In the third step, a change with time in the light emission amount of the biological sample is examined based on a plurality of light emission images obtained by imaging. Specifically, the light emission amount is digitized. For example, for each luminescent image, data corresponding to the brightness in a specific region of the luminescent image (hereinafter referred to as luminance data) is extracted. The light emission amount is, for example, a value corresponding to luminance data or a value corresponding to an integrated value of luminance data in a specific section. It is considered that the luminescence amount or luminance data obtained for the region corresponding to the biological sample in the luminescent image reflects the amount of luciferase in the biological sample. Normally, this area has a size corresponding to a plurality of pixels of the image sensor. Therefore, usually, a plurality of luminance data are extracted from the previous area and their sum is obtained. As a means for extracting this data, for example, a computer program for image analysis can be used. Using this program, for example, a specific area of one of the luminescent images is specified, and luminance data in the previous area is extracted from the luminescent image and other luminescent images. The area from which the luminance data is extracted can be arbitrarily selected from the image. Depending on the purpose of the study, for example, a region corresponding to one or more cells can be selected. For example, it is possible to select a region corresponding to a cell that takes a specific form or is in a specific state in the cell cycle in the first or second luminescent image. You can select the area corresponding to a single cell and extract the luminance data of that area, or you can select multiple areas corresponding to multiple cells and add the luminance data of those areas together. Also good.

  The biological sample may further contain a factor for adjusting the amount of luciferase luminescence in the biological sample.

  Such a factor may be a factor that regulates the amount of luciferase. For example, this factor may be adjusted so that the amount of luciferase is always constant during imaging, or may be adjusted to increase or decrease along an arbitrary pattern. For example, when a luciferase gene is introduced into a biological sample and luciferase is expressed from this gene, an arbitrary promoter can be placed upstream of this gene. As such a promoter, for example, a promoter that always promotes expression, a promoter that promotes expression in response to an external stimulus, or a promoter that promotes expression in accordance with an intracellular state can be used. For example, a promoter of a clock gene can be used, and for example, a promoter of mPer2 can be used. Such a promoter can be selected according to the purpose of research.

  Alternatively, such a factor may be a factor that regulates the activity of luciferase. Such a factor is, for example, a cofactor essential for a luminescence reaction catalyzed by luciferase. Examples of such cofactors are ATP and Mg ions.

  The luciferase may be fused with another protein (referred to as “first protein”) in the biological sample. That is, luciferase may be used as a label for the first protein in the biological sample. In this case, the amount of luciferase reflects the expression level of the first protein in the biological sample. The first protein is preferably a marker that indicates the state of a cell. That is, the first protein is preferably a protein that is endogenously expressed in the cell, and the expression level of which varies depending on the state of the cell. For example, the first protein may be a marker associated with a selective proteolytic reaction, a marker consisting of all or part of a protein exhibiting protein interaction, a marker consisting of all or part of a ligand detection protein, an embryo development It is preferably a marker, a neuronal differentiation marker, or an ES cell differentiation marker. In particular, the first protein is preferably a marker associated with a selective proteolytic reaction. By observing the expression state of such a marker as the amount of luciferase emitted, the state of the cell can be estimated. Introduction of the first protein labeled with luciferase into the biological sample can be carried out in the same manner as described above.

  The biological sample may further contain a labeled second protein that is different from the first protein. As the label, a fluorescent molecule or a luminescent molecule can be used. For example, fluorescent proteins such as GFP and YFP can be used. In this case, the presence or absence of the second protein and its localization can be confirmed in the image acquired in the second step. As the second protein, for example, a protein for which a function in a biological sample is to be studied is selected. When the first protein and the second protein are simultaneously introduced into the biological sample, the state of the cell is estimated based on the amount of luciferase luminescence, and the behavior of the second protein in that state can be observed. It becomes possible. Alternatively, a marker different from the first protein is selected as the second protein. In this case, two criteria, the first protein and the second protein, can be used to estimate the state of the cell.

  Furthermore, the biological sample may contain one or more proteins different from the first protein and the second protein. Those proteins may be labeled.

  Luciferin may be introduced into the biological sample in the first step. Further, the luciferin regenerating enzyme may be introduced into the biological sample in the second step.

  As a conventional technique, there is a method in which a luciferase and a luciferin regenerating enzyme are simultaneously expressed in a cell and the amount of luminescence of the whole cell is measured with a luminometer or the like. In this method, luciferin regenerating enzyme is mainly used for the purpose of enhancing the amount of luminescence in the short term. Further, in this method, since the amount of luminescence of the entire cell is measured, the relationship between the cell state and the luminescence state cannot be examined for each cell.

  On the other hand, in the analysis method according to the present invention, the state of the biological sample or a part thereof is associated with the brightness of the region corresponding to each biological sample or part thereof in the luminescent image. For example, a change in the amount of luciferase luminescence is associated with a circadian rhythm controlled by a clock gene. Similarly, fluctuations in the amount of luciferase luminescence are associated with, for example, the state of the cell cycle, embryogenesis, neuronal differentiation or ES cell differentiation. By associating such association with information such as the morphology of the cell obtained from the luminescence image and the behavior of another protein in the cell, useful information can be obtained. For example, it is possible to study what form a cell takes at a certain stage of the circadian rhythm, or what behavior a particular protein takes within the cell. According to the method according to the embodiment, such research can be performed in detail with high sensitivity over a long period of time. In particular, it is possible to shorten the exposure time required to capture a luminescent image, to observe for a longer time than before, and to perform temporal observation with higher time resolution than before.

  The second embodiment of the present invention is a luciferin regenerating enzyme protein.

  The luciferin regenerating enzyme protein according to the embodiment is derived from, for example, an organism belonging to the arthropoda elegans Coleoptera firefly family. That is, the luciferin-regenerating enzyme protein according to the embodiment is a luciferin-regenerating enzyme protein obtained from an organism belonging to the arthropoda genus Coleoptera firefly family or a variant thereof.

  A specific example of the luciferin regenerating enzyme protein according to the embodiment has the amino acid sequence set forth in SEQ ID NO: 1. This luciferin-regenerating enzyme protein was newly obtained from Japanese-made Chromadobotaru (Pyrocoelia fumosa). Regarding this protein, no protein homologous of 69% or more has been registered in a known protein database search.

This amino acid sequence is as follows.
MGPVVEKIEALGNFMVGEGPHWDHQTQALYFVDILGKTIHKYIPSLEKHTSLKVDQAPSFIIPVTGSSDRFVISLEGDIGILTWNGIDSAPTSIKIVGTVDSEPGNENNRINDGKADPLGNLWAGTMAIDADLPVGPVRGTLYSLTPDKQFKSHAKNIAISNGLAWSKDLKKMYYIDSAKRGIDQYDFDASTLSISNCQPLFTFEKHQIPGYPDGQTIDADGNLWVAVFQGNRVIKISTNKPEMLLDTIEIPDHQVTSVAFGGPNLDVLYVTSAGKPLHCSPGSSIYGHLYKVTGLGVNGLPGDTVNI (SEQ ID NO: 1).

  Another example of the luciferin regenerating enzyme protein according to the embodiment is a variant of a protein having the amino acid sequence set forth in SEQ ID NO: 1. This mutant has, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% of the amino acid sequence between the wild-type amino acid sequence As described above, it has a homology of 97% or more, 98% or more, or 99% or more, and has activity as a luciferin regenerating enzyme.

  The luciferin regenerating enzyme protein according to the embodiment can be used in the analysis method according to the embodiment.

  The third embodiment of the present invention is a nucleic acid containing a luciferin regenerating enzyme gene.

  The luciferin regenerating enzyme gene according to the embodiment is derived from, for example, an organism belonging to the arthropoda elegans Coleoptera firefly family. That is, it is a luciferin-regenerating enzyme gene obtained from an organism belonging to the arthropoda genus Coleoptera firefly family or a mutant thereof.

  Specific examples of the nucleic acid according to the embodiment include a luciferin regenerating enzyme gene having the base sequence set forth in SEQ ID NO: 2. This luciferin regenerating enzyme gene was newly obtained from Japanese-made Chromadobotaru (Pyrocoelia fumosa) and encodes a protein having the amino acid sequence of SEQ ID NO: 1.

This base sequence is as follows.
(SEQ ID NO: 2).

  Another example of the nucleic acid according to the embodiment is a variant of the nucleic acid having the base sequence set forth in SEQ ID NO: 2. For example, the base sequence of this mutant is 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more with a wild-type amino acid, A protein having a homology of 97% or more, 98% or more, or 99% or more, and the protein encoded by this mutant has activity as a luciferin regenerating enzyme.

  The nucleic acid according to the embodiment can be used in the analysis method according to the embodiment.

[Example 1: Cloning of LRE protein gene derived from Japanese firefly family Chromadobotaru]
The LRE protein gene derived from the Japanese firefly family Chromadobotaru was cloned. Briefly, cDNA and other libraries were prepared based on total RNA extracted from this organism, and the gene was cloned using PCR as a template.

1. Preparation of cDNA library A 0.009 g light emitter was cut out from a single larvae of Chromadobotaru collected around Mt. Mitake in Tokyo in August 2007. The collected light emitter and 1 mL of total RNA extraction reagent TRIzol Reagent (Invitrogen) were placed in a Lysing Matrix D tube (MP-Biomedicals). This tube contains beads for tissue and cell homogenization. This tube was attached to a tissue cell crushing device FastPrep 24 (MP-Biomedicals), and a firefly luminescence device was crushed in a reagent under conditions of a vibration speed of 6.5 m / s and a vibration time of 45 seconds. After completion, the tube was removed from the crusher and placed on ice for 30 minutes. Thereafter, crushing was performed again under the same conditions.

  Next, according to the instructions of the total RNA extraction reagent TRIzol Reagent, the total RNA was separated and purified from the disrupted solution. 100 μl of the obtained mRNA solution was concentrated by precipitation by ethanol precipitation. Next, a full-length cDNA was synthesized from the precipitated and concentrated total RNA using a full-length cDNA synthesis reagent GeneRacer (Invitrogen) according to the manual. 20 μl of the obtained cDNA solution was used as a firefly full-length cDNA library for the following gene experiments.

2. 2. Identification of 5 ′ end side of firefly LRE gene 2-1. Preparation of Primer for Rapid Amplification of cDNA End (RACE) Method Cloning of the LRE gene was performed by the Polymerase chain reaction (PCR) method. Primers used in the PCR method were prepared as follows based on the amino acid sequences of LRE genes derived from known closely related organisms.

  In order to confirm amino acid regions that are well conserved in various firefly LREs, the amino acid sequences of three types of already-disclosed firefly LREs are compared using sequence information analysis software DNASIS Pro (Hitachi Software Engineering Co., Ltd.) did. The amino acid sequences of closely related organisms used for comparison are Photinus pyralis (registration number: AB062786), Lucio cruciata (registration number: AB072448) and Luciola lateralis (registration number: AB072447).

  As a result of comparison, the amino acids of VTGLGLGVKG (SEQ ID NO: 3) and NDGGGAD (SEQ ID NO: 4) in the vicinity of the C-terminus of LRE The sequence was found to be well conserved. Base sequences are predicted from codons encoding these two amino acid sequences, and 16 types (SEQ ID NOs: 5 to 20) and 4 types (SEQ ID NOs: 21 to 24) of LRE-specific mixed primers used for 5 ′ terminal RACE PCR are designed. did. The synthesis of these primers was outsourced to Life Technologies Japan.

The names and sequences of these primers are as follows (Y and N in the primer sequence indicate mixed bases):
LRE-R1-AA (5′-CCYTTNACNCCNARNCCTGTTAC-3 ′: SEQ ID NO: 5),
LRE-R1-TA (5′-CCYTTNACNCCNARNCCTGTAAC-3 ′: SEQ ID NO: 6),
LRE-R1-GA (5′-CCYTTNACNCCNARNCCTGTCAC-3 ′: SEQ ID NO: 7),
LRE-R1-CA (5′-CCYTTNACNCCNARNCCTGTGAC-3 ′: SEQ ID NO: 8),
LRE-R1-AT (5′-CCYTTNACNCCNARNCCAGTTAC-3 ′: SEQ ID NO: 9),
LRE-R1-TT (5′-CCYTTNACNCCNARNCCAGTAAC-3 ′: SEQ ID NO: 10),
LRE-R1-GT (5′-CCYTTNACNCCNARNCCAGTCAC-3 ′: SEQ ID NO: 11),
LRE-R1-CT (5′-CCYTTNACNCCNARNCCAGTGAC-3 ′: SEQ ID NO: 12),
LRE-R1-AC (5′-CCYTTNACNCCNARNCCGGTTAC-3 ′: SEQ ID NO: 13),
LRE-R1-TC (5′-CCYTTNACNCCNARNCCGGTAAC-3 ′: SEQ ID NO: 14),
LRE-R1-GC (5′-CCYTTNACNCCNARNCCGGTCAC-3 ′: SEQ ID NO: 15),
LRE-R1-CC (5′-CCYTTNACNCCNARNCCGGTGAC-3 ′: SEQ ID NO: 16),
LRE-R1-AG (5′-CCYTTNACNCCNARNCCCGTTAC-3 ′: SEQ ID NO: 17),
LRE-R1-TG (5′-CCYTTNACNCCNARNCCCGTAAC-3 ′: SEQ ID NO: 18),
LRE-R1-GG (5′-CCYTTNACNCCNARNCCCGTCAC-3 ′: SEQ ID NO: 19),
LRE-R1-CG (5′-CCYTTNACNCCNARNCCCGTGAC-3 ′: SEQ ID NO: 20),
LRE-R2-AA (5′-TCnGCYTTnCCATCATT-3 ′: SEQ ID NO: 21),
LRE-R2-GA (5′-TCnGCYTTnCCGTCATT-3 ′: SEQ ID NO: 22),
LRE-R2-AG (5′-TCnGCYTTnCCATCGTT-3 ′: SEQ ID NO: 23) and LRE-R2-GG (5′-TCnGCYTTnCCGTCGTT-3 ′: SEQ ID NO: 24).

2-2.5′-RACE PCR cloning of the 5 ′ end of the firefly LRE gene Using the firefly full-length cDNA library prepared as described above as a template, V-T-G-L- 16 types of primers prepared based on GVKG (SEQ ID NO: 3) and 4 types of specific mixed primers prepared based on NDGGAD (SEQ ID NO: 4) GeneRacer 5 'Primer (5'-CGA CTG GAG CAC GAG GAC ACT GA-3': SEQ ID NO: 25) and GeneRacer 5 'Nested Primer (5'-GGA CAC TGA CAT GGA CTG AAG GAG TA-3 ′: SEQ ID NO: 26) and 5′-RACE PCR and nested PCR I went. The GeneRacer 5 ′ Primer and the GeneRacer 5 ′ Nested Primer included in the full-length cDNA synthesis reagent GeneRacer kit (Invitrogen) were used. In order to efficiently amplify the LRE gene by 5′-RACE PCR, a nested PCR was performed in which the gene amplified once by PCR was used as a template and the gene was amplified more specifically by the inner primer pair. PCR was performed using polymerase ExTaq (Takara Bio Inc.) according to the manual.

  Using the 16 primers prepared based on VTGLGLGVKG (SEQ ID NO: 3) and GeneRacer 5 'Primer, the first 5'-RACE PCR was performed according to the manual. As a result, clear gene amplification could not be confirmed. A second nested PCR was carried out using this PCR product as a template.

  Nested PCR was performed using four types of primers prepared based on NDGGAD (SEQ ID NO: 4) and GeneRacer 5'Nested Primer. As a result, the product of PCR performed in combination with LRE-R1-GA (SEQ ID NO: 7) or LRE-R1-CT (SEQ ID NO: 12) and GeneRacer 5 ′ Primer was used as a template, and LRE-R2-AA (SEQ ID NO: 21). ) And GeneRacer 5 ′ Nested Primer was used, and the gene was efficiently amplified in the vicinity of about 0.5 kbp by nested PCR.

Determination of the base sequence of the gene amplified by 2-3.5′-RACE In order to read the base sequence of the gene amplified by 5′-RACE, the PCR product was purified by gel extraction, subcloning and direct sequencing. Details are shown below.

  A gene of about 0.5 kbp was recovered using a gel extraction technique. Gel extraction was performed according to the manual using Wizard SV Gel and PCR Clean-Up System (Promega Corporation). Subcloning of the PCR product extracted from the gel was performed using the TA cloning technique. TA cloning was performed using pGEM-T Easy Vector System (Promega Corporation) according to the manual. Thereafter, this vector DNA was transformed into E. coli (DH5α strain), and an insert positive colony was selected using a blue / white screening technique. The selected colony was subjected to direct colony PCR to confirm that the gene was introduced. For direct colony PCR, M13-F (−29) Primer (5′-CAC GAC GTT GTA AAA CGA C-3 ′: SEQ ID NO: 27) and M13 Reverse (5′-GGA TAA CAA TTT CAC AGG-3 ′: A primer pair consisting of SEQ ID NO: 28) was used.

  For the PCR reaction solution in which amplification was confirmed, the base sequence of the gene was determined using the direct sequencing method. Excess dNTPs and primers contained in the PCR reaction solution were removed using a PCR product purification kit ExoSAP-IT (GE Healthcare Bioscience) to prepare a template for PCR direct sequencing. Using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), a sequencing reaction solution containing this template was prepared, and a sequencing reaction was performed using a thermal cycler. PCR product purification and sequencing were performed according to the respective manuals. After the sequencing reaction, the reaction product was purified as follows. 2.5 times volume of 100% ethanol was added to the reaction solution, and nucleic acid was precipitated using a centrifuge. Next, after removing the supernatant, 70% ethanol was added to wash the precipitate, and the nucleic acid was precipitated using a centrifuge. Finally, after removing the upper fine, the precipitate was dried. To the purified precipitate, 15 μl of Hi-Di Formatide (Applied Biosystems) was added and dissolved. This solution was heat-denatured at 94 ° C. for 2 minutes and further rapidly cooled on ice to prepare a sample for determining the base sequence. The base sequence of this sample was read using an Applied Biosystems 3130xl genetic analyzer (Applied Biosystems). The base sequence analysis method was performed according to the manual.

The gene sequence obtained by sequencing is as follows:
(SEQ ID NO: 29).

  This sequence was subjected to homology search using a blastx search provided by National Center for Biotechnology Information (hereinafter abbreviated as NCBI), and confirmed to show homology with the nucleotide sequence of known LRE. The base sequence obtained by the above experiment and analysis was determined to be on the 5 'end side of the novel LRE gene.

3. 3 'Race PCR of LRE gene and acquisition of full-length cDNA 3-1.3' Design of primers used for 'Race PCR' Based on the sequence of the untranslated region on the 5 'end side of LRE gene obtained in 5' Race PCR experiments Thus, two primers used for 3′RACE were prepared (5′-AGGCAGTCAATTCATATCAGAC-3 ′: SEQ ID NO: 30 and 5′-AGGCAGCACATCTAATTCAGACCAAATAGGGG-3 ′: SEQ ID NO: 31). Primer synthesis was outsourced to Life Technologies Japan.

3-2. 3 'Race PCR for obtaining full-length cDNA of firefly LRE gene
Using the firefly full-length cDNA library prepared as described above as a template, two primers prepared from the base sequence of the 5′-terminal untranslated region of the target firefly LRE, GeneRacer 3 ′ Primer (SEQ ID NO: 32) and GeneRacer 3 3'-RACE PCR and 3'-Nested PCR were performed using 'Nested Primer (SEQ ID NO: 33). GeneRacer 3 ′ Primer and GeneRacer 3 ′ Nested Primer used were those included in the full-length cDNA synthesis reagent GeneRacer kit (Invitrogen). PCR was performed using polymerase ExTaq (Takara Bio Inc.) according to the manual.

  The LRE gene was amplified using a primer pair consisting of a primer (SEQ ID NO: 30) prepared from the base sequence of the 5 'terminal side untranslated region and GeneRacer 3' Primer (SEQ ID NO: 32). Using this PCR product as a template, 3'-Nested PCR was performed using a primer (SEQ ID NO: 31) prepared from the base sequence of the 5'-terminal untranslated region and GeneRacer 3 'Nested Primer (SEQ ID NO: 33). As a result, it was confirmed that the gene was efficiently amplified in the vicinity of about 1.2 kbp.

Determination of the base sequence of the gene amplified with 3-3.3′-Race In order to read the base sequence of the gene amplified with 3′-RACE, purification of the PCR product by gel extraction, subcloning and direct sequencing were performed. Details are shown below.

  A gene fragment was recovered by using a gel extraction technique for a band in which the gene was efficiently amplified in the vicinity of about 1.2 kbp. Gel extraction was performed according to the manual using Wizard SV Gel and PCR Clean-Up System (Promega Corporation). Subcloning of the PCR product extracted from the gel was performed using the TA cloning technique. TA cloning was performed using pGEM-T Easy Vector System (Promega Corporation) according to the manual. Thereafter, this vector DNA was transformed into E. coli (DH5α strain), and an insert positive colony was selected using a blue / white screening technique. The selected colony was subjected to direct colony PCR to confirm that the gene was introduced. For direct colony PCR, a primer pair consisting of M13-F (-29) Primer and M13 Reverse was used.

  For the PCR reaction solution in which amplification was confirmed, the base sequence of the gene was determined using the direct sequencing method. Excess dNTPs and primers contained in the PCR reaction solution were removed using a PCR product purification kit ExoSAP-IT (GE Healthcare Bioscience) to prepare a template for PCR direct sequencing. Using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), a sequencing reaction solution containing this template was prepared, and a sequencing reaction was performed using a thermal cycler. Primers used for sequencing were vector primers or gene-specific primers. PCR product purification and sequencing were performed according to the respective manuals.

The full length LRE gene was obtained by sequencing. The base sequence was as follows:
(SEQ ID NO: 34).

  When this base sequence (SEQ ID NO: 34) was translated into an amino acid, it was predicted that a PCR error would be present at the position of R49. Was introduced. Mutation was introduced based on the description in the literature (Asako Sawano and Atsushi Miyawaki, Nucleic Acids Research, 2000, Vol. 28, No. 16). The sequence translated into the base sequence (SEQ ID NO: 2) or amino acid (SEQ ID NO: 1) was subjected to homology search using blastx or blastp search provided by NCBI. It was confirmed that each search showed homology with the base sequence of known firefly LRE. The base sequence obtained by the above experiment and analysis was determined to be the full-length cDNA sequence of the novel firefly LRE gene.

  Hereinafter, this new LRE is referred to as a chromatobotanic LRE.

[Example 2: Purification of chromatobotan LRE]
The LRE gene was introduced into pRSET-B vector (Invitrogen) for protein expression in E. coli. The gene expression vector was constructed according to standard methods. Thereafter, the constructed gene expression vector was transformed into Escherichia coli to express the protein, and then the protein was purified using a nickel affinity purification method. The luminescence enhancing effect of the purified protein was measured. Details are shown below.

1. Introduction of a novel firefly LRE gene into a gene expression vector In order to introduce the LRE gene between the restriction enzyme sites BamHI and EcoRI of the pRSET-B vector, a primer (5 ′ containing a restriction enzyme BamHI recognition sequence GGATCC in front of the initiation codon -GCGgatccCaccATGGGACCTGTTGTAGAAAAAATCG-3 ': SEQ ID NO: 37) and a primer (5'-CGGgaattcTCAAATGTTCACTGTATCCTCTGGC-3': SEQ ID NO: 38) containing a stop codon and a restriction enzyme EcoRI recognition sequence GAATTC behind it. Using this primer pair, a fragment containing the above-mentioned restriction enzyme recognition sites at both ends of the LRE gene was amplified. PCR was performed using polymerase ExTaq (Takara Bio Inc.) according to the manual. The PCR product containing the restriction enzyme site and the pRSET-B vector were treated with a restriction enzyme and introduced into the restriction enzyme site BamHI-EcoRI of the pRSET-B vector using a ligation kit (Takara Bio Inc.). The obtained ligation sample was transformed into E. coli JM109 (DE3) strain to form colonies. From this, E. coli JM109 expressing the LRE gene was extracted.

2. Purification of LRE protein A colony of E. coli JM109 expressing the LRE gene was picked up and pre-cultured at 37 ° C for 24 hours using 2 ml scale LB medium. The pre-cultured culture solution was transferred to 30 mL of LB medium and cultured at 26 ° C. for 24 hours. After culturing, the cells were collected by centrifugation, suspended again in Lysis Buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl) containing protease inhibitor complete (Roche) and sonicated. One tablet of protease inhibitor complete (Roche) was added to 30 mL of Lysis Buffer. The cell disruption solution was centrifuged (10000 g, 10 minutes, 4 ° C.), the sediment was removed, and the supernatant was recovered. Affinity purification was performed using 1 mL of Ni resin (Roche). All operations until the entire amount of the supernatant passed through the column were performed at 4 ° C. Lysis buffer containing 400 mM imidazole was added to elute LRE. The eluted sample was filtered through a gel filtration column PD-10 (GE Healthcare) and desalted. The sample after desalting was concentrated by ultrafiltration (10 k).

3. Measurement of Luminescence Enhancement Effect Using a LumiFl SpectroCapture (ATTO) as an apparatus for measurement, a change in luminescence amount with time was measured under an experimental condition of 12 hours at a measurement interval of 2 minutes. For the measurement, A solution containing 3.3 μg of LRE and purified CBR luciferase (Promega) and B solution containing the substrate were mixed immediately before the measurement. Solution A is 50 μL of Buffer A (50 mM Tris-HCl (pH 8.0), 20 mM NaCl) containing 8 mM D-cysteine, 4 mM ATP, 4 mM MgSO ₄ . Solution B is 50 μL of Buffer A (50 mM Tris-HCl (pH 8.0), 20 mM NaCl) containing 2 mM D-luciferin. As a control, solution C containing no LRE and solution D containing no luciferase were prepared, and each solution B containing a substrate was mixed immediately before the measurement to measure the amount of luminescence. The measurement results are shown in FIGS.

  FIG. 1 shows changes over time in the luminescence amounts of Liquid A + B Liquid (CBR + Kuromado-LRE), Liquid C + B Liquid (CBR), and Liquid D + B Liquid (Kuromado-LRE). It is shown in 2. From FIG. 1, it can be seen that the luminescence reaction to which LRE was added showed a more stable luminescence reaction than the luminescence reaction to which LRE was not added. FIG. 2 shows that the addition of the LRE enhances the luminescence activity of the known luciferase by at least 1.3 times or more in the integrated value for 12 hours. Further, the LRE alone does not emit light.

[Example 3: Intracellular luminescence enhancement effect of novel LRE]
In order to express the firefly LRE gene in a mammalian cell, the gene is introduced into a pcDNA3.1 (+) vector (Invitrogen). Construction of the gene expression vector is carried out according to standard methods. Thereafter, the constructed gene expression vector is introduced into a mammalian cell to express the protein, and then the effect of enhancing luminescence in the cell is measured. Details are shown below.

1. Introduction of a novel firefly LRE gene into a gene expression vector In order to introduce the LRE gene between the restriction enzyme sites BamHI and EcoRI of the pcDNA3.1 (+) vector, the restriction enzyme BamHI recognition sequence GGATCC and the Kozak sequence are preceded by the start codon. And a primer (SEQ ID NO: 38) containing a stop codon followed by the restriction enzyme EcoRI recognition sequence GAATTC. Using this primer pair, a fragment containing the above-mentioned restriction enzyme recognition sites at both ends of the luciferase gene is amplified. For PCR, polymerase KOD-Plus- (Toyobo Co., Ltd.) is used according to the manual.

10 × PCR Buffer with final concentration of 1 ×, final concentration of 0.2 mM dNTP Mixture (2.5 mM each), final concentration of 1.0 mM MgSO ₄ , final concentration of 0.02 U / μl TOYOBO KOD− Plus- (1 U / μl), a 20 μl PCR reaction solution containing a primer pair each having a final concentration of 0.3 μM was prepared, and the LRE gene (SEQ ID NO: 2) having no BamHI and EcoRI recognition sequences was used as a template. Add 0.4 μl of solution. In the PCR reaction, after heat denaturation at 94 ° C. for 2 minutes, 94 cycles of 30 ° C., 55 ° C. for 30 seconds and 68 ° C. for 2 minutes are repeated 30 cycles, and finally, an extension reaction at 68 ° C. for 5 minutes is performed. After the PCR reaction, 1 μl of the PCR reaction solution is electrophoresed using a 1% TAE agarose gel. After ethidium bromide staining, the amplified gene band is observed under ultraviolet irradiation. When gene amplification is observed, this PCR reaction solution is precipitated and concentrated by an ordinary ethanol precipitation method, 4 μl of restriction enzyme treatment 10 × H Buffer, restriction enzyme BamHI (Toyobo Co., Ltd.) and restriction enzyme EcoRI (Toyobo Co., Ltd.) 2) each and 32 μl of sterilized deionized water are dissolved, and the mixture is incubated at 37 ° C. for 2 hours for restriction enzyme treatment. Thereafter, the reaction solution is precipitated and concentrated by an ethanol precipitation method and then dissolved in sterilized deionized water. This solution is electrophoresed on a 1% TAE agarose gel and ethidium bromide staining is performed. A gel containing a DNA band identified under ultraviolet irradiation is cut out using a knife. DNA is extracted from this excised gel using Wizard (R) SV Gel and PCR Clean-Up System (Promega). These operations are performed according to the manual. Thereafter, using Ligation Pack (Nippon Gene) according to the manual, the extracted DNA is introduced into the pcDNA3.1 (+) vector previously treated with restriction enzyme BamHI and restriction enzyme EcoRI by the same method. This vector DNA is transformed into DH5α strain to form colonies.

Direct colony PCR is performed using the obtained colony as a template to amplify the LRE gene introduced into the pcDNA3.1 (+) vector. Direct colony PCR was performed using T7 promoter Primer (5′-TAA TAC GAC TCA CTA TAG GG-3 ′: SEQ ID NO: 39) and T7 Reverse Primer (5′-TAG AAG GCA CAG TCG AGG C-3 ′: SEQ ID NO: 40). The primer pairs are used. Final concentration of 10 × Ex Taq Buffer (20 mM Mg ²⁺ plus), final concentration of 0.2 mM each dNTP Mixture (2.5 mM each), final concentration 0.05 U / μl TaKaRa Ex Taq (5 U / μl) and 10 μl of PCR reaction solution containing primers each having a final concentration of 0.2 μM are prepared, and a small amount of E. coli colony is added thereto as a template. In the PCR reaction, after heat denaturation at 94 ° C. for 2 minutes, 94 cycles of 30 ° C., 50 ° C. for 30 seconds and 72 ° C. for 2 minutes are repeated 25 cycles, and finally, extension reaction at 72 ° C. for 5 minutes is performed. After the PCR reaction, 1 μl of the PCR reaction solution is electrophoresed using a 1% TAE agarose gel. After ethidium bromide staining, the amplified gene band is observed under ultraviolet irradiation.

  For the PCR reaction solution in which amplification has been confirmed, the base sequence of the gene is determined using the direct sequencing method. Using PCR product purification kit ExoSAP-IT, excess dNTP and primers contained in the PCR reaction solution are removed, and a template for PCR direct sequencing is prepared. A sequencing reaction solution containing this template is prepared using BigDye Terminator v3.1 Cycle Sequencing Kit, and a sequencing reaction is performed using a thermal cycler. For the sequence, a vector primer or a gene-specific primer is used. PCR product purification and sequencing are performed according to the respective manuals. After the sequencing reaction, the reaction product is purified as follows. Add 2.5 volumes of 100% ethanol to the reaction solution and precipitate the nucleic acid using a centrifuge. Next, after removing the supernatant, 70% ethanol is added to wash the precipitate, and the nucleic acid is precipitated using a centrifuge. Finally, after removing the upper fine, the precipitate is dried. Add 15 μl of Hi-Di Formatide (Applied Biosystems) to the purified precipitate and dissolve. This solution is heat denatured at 94 ° C. for 2 minutes and further rapidly cooled on ice to prepare a sample for determining the base sequence. The base sequence of this sample is read using an Applied Biosystems 3130xl genetic analyzer to confirm that it has been normally introduced into the gene expression vector pcDNA3.1 (+).

2. Introduction of novel firefly LRE gene into mammalian cells and measurement of luminescence enhancement effect In order to measure the luminescence enhancement effect of LRE in mammalian cells, pGL4-control vector (Promega) and the LRE gene were co-expressed. A luminescent activity measurement is performed. Specifically, a pGL4-control vector (0.5 μg) and a pcDNA3.1 (+) vector (2.5 μg) containing the LRE gene are introduced into mammalian cells (condition 2). For comparison, a plasmid into which a pGL4-control vector (0.5 μg) and a pcDNA3.1 (+) vector (2.5 μg) have been introduced is prepared (Condition 1). The reason why the pcDNA3.1 (+) vector was added under the condition 2 is to make the transformation efficiency of the pGL4-control vector into cells uniform.

  In each of the conditions 1 and 2, the gene is introduced into HeLa cells seeded in a 35 mm dish using the gene introduction reagent Lipofectamine 2000 (Invitrogen). The method is carried out according to the manual. After 24 hours, the cells are washed with PBS. 2 ml each of 500 μM D-luciferin / DMEM HEPES medium (Invitrogen) was added to each dish for luminescence measurement, and was continuously used for 20 hours at 36 ° C., luminescence measurement time 10 seconds, luminescence observation interval 3 minutes using chronos (ATTO). The light emission intensity is measured under the measurement conditions. The expected results from this measurement are shown in FIGS.

  FIG. 3 shows a value obtained by integrating the light emission amount for a certain time. The vertical axis represents the light emission amount. As shown in FIG. 3, a higher amount of luminescence is obtained in condition 2 than in condition 1, and the LRE according to the embodiment is expected to have an effect of enhancing the luminescence activity of known luciferase.

  FIG. 4 shows the change in the amount of light emitted over time. The vertical axis represents the light emission amount. As shown in FIG. 4, when LRE is not expressed, the amount of luminescence changes at a very low level, whereas when LRE is expressed, the amount of luminescence is expected to be very high.

  From the above results, it is expected that the combination of the known luciferase and the LRE according to the embodiment continuously exerts the luminescence enhancing effect even in living cells.

[Example 4: Comparison of chromatobotan LRE and known LRE with respect to luminescence enhancement effect in cells]
The effect of enhancing luminescence in cells is compared between the chromatobotanic LRE and the known LRE. Known LREs include Heikebotaru LRE (Luciola cruciata_LRE (AB072448): SEQ ID NO: 42) disclosed in Japanese Patent No. 04158956 and Genji Botanical LRE (Luciola lateris_LRE (Sequence No. AB0743) disclosed in Japanese Patent No. 03891323): To do.

1. Preparation of Expression Vector In the same manner as in Example 3 above, Heike firefly LRE and Genji firefly LRE are introduced into pcDNA3.1 (+) vector (Invitrogen) to prepare two types of known LRE mammalian expression vectors.

  An expression vector containing the gene for Lucinase derived from Photinus pyralis (Luc2-pGL4 vector (Promega)) (SEQ ID NO: 47) and the gene for Lucifera accensa (SEQ ID NO: 44 and 45) is prepared as follows. That is, a Kozak sequence is added immediately before the start codon of each luciferase gene, and inserted between the SgfI and PmeI sites of the multicloning site of pF9A CMV hRLuc neo Flexi vector (Promega).

2. Measurement of luminescence enhancement effect of new LRE and known LRE in cells Each LRE is expressed in cells in combination with luciferase as follows:
(Condition 1) A combination of a chromatobotan LRE and a highly luminescent Lucidina accensa luciferase;
(Condition 2) A combination of Heike firefly-derived LRE and Photinus pyralis-derived luciferase (Luc2-pGL4 vector (Promega)); and (Condition 3) A combination of Genji firefly-derived LRE and Photinus pyralis-derived luciferase (Luc2-pGL4 vector (Promega)) .

  In each combination, a vector containing luciferase (0.5 μg) and each LRE gene pcDNA3.1 (+) vector (2.5 μg) are introduced into HeLa cells. Specifically, the gene is introduced into HeLa cells seeded in a 35 mm dish using a gene introduction reagent FuGENE HD (Promega) according to the manual. After 24 hours, the cells are washed with PBS. For luminescence measurement, 2 ml of 500 μM D-luciferin / DMEM HEPES medium (Invitrogen) was added to each dish, and the luminescence measurement time was 36 ° C., luminescence measurement time 10 seconds, luminescence observation interval 48 minutes at 3 minutes. The amount of luminescence is measured under the condition of continuous time measurement. The expected results from this measurement are shown in FIGS.

  As shown in FIG. 5, when a known luciferase is combined with a chromatobotan LRE (Condition 1), it is expected that the amount of luminescence is much higher than when combined with a known LRE (Conditions 2 and 3). Is done.

  In addition, as shown in FIG. 6, when the known luciferase is combined with the chromatobotan LRE (condition 1), the amount of luminescence is continuously higher than when combined with the known LRE (conditions 2 and 3). It is expected that.

[Example 5: Analysis of clock gene using LRE derived from Chromadota firefly]
Organisms have biological clocks that control rhythms of about 24 hours, and various clock gene promoter assays have been performed to understand them. In the analysis of clock genes using bioluminescence, it is indispensable not only to emit light stably, but also to smoothly attenuate the light emission according to the rhythm of the clock gene. In the luminescence measurement using the chromatobotanic LRE, not only the effect of increasing the luminescence amount but also the effect on the stabilization of phase and amplitude required for clock gene analysis and the like are compared.

1. Preparation of expression vector Using clock oscillation gene mPer2 gene and firefly luciferase reporter vector pGL4.12 Vector (Promega) in which proteolytic sequences hCL1 and hPEST are arranged on the C-terminal side of luciferase gene, Perform reporter analysis. That is, a reporter gene mPer2 :: pGL4.12 Vector in which a luciferase is linked to the promoter of the clock gene mPer2 (including recognition sites for KpnI and XhoI) (SEQ ID NO: 48) is prepared. Specifically, an expression vector for luminescence analysis is prepared by introducing the mPer2 gene between NheI and XhoI, which are vector restriction enzyme sites, using the same method as in Example 4.

2. Luminescent promoter measurement using chromatobotan LRE The mPer2 :: pGL4.12 Vector prepared as described above is used in the following combinations:
(1) Combination of mPer2 :: pGL4.12 Vector (0.5 μg) and pcDNA3.1 (+) vector (2.5 μg); and (2) mPer2 :: pGL4.12 Vector (0.5 μg) and chromatobotan Combination with the derived LRE gene pcDNA3.1 (+) vector (2.5 μg).
The reason why the pcDNA3.1 (+) vector was used in (1) is to make the transformation efficiency of mPer2 :: pGL4.12 Vector into cells uniform.

  Using the combination of the above vectors, the gene is introduced into NIH3T3 cells seeded in a 35 mm dish using the gene introduction reagent Lipofectamine 2000 (Invitrogen) according to the manual. At 24 hours after gene transfer, clock gene transmission is synchronized by 100 nM Dexamezone stimulation. Luminescence measurement was performed by adding a final concentration of 500 μMD-Luciferin to culture medium DMEM-HEPES High Glucose (Gibco 21063-029) and using chronos (ATTO) at 36 ° C., luminescence measurement time of 10 seconds, and luminescence observation interval of 3 minutes. The emission intensity is measured under the condition of continuous time measurement. The expected results from this measurement are shown in FIGS. 7a and 7b.

  7a and 7b show the change in the amount of light emitted over time. The vertical axis represents the light emission amount. FIG. 7a shows the change in the expression level from 123 hours after the start of observation, and FIG. 7b shows the change in the expression level from 40 hours to 123 hours after the start of observation, which is a section surrounded by the broken line in FIG. 7a. . From these figures, in the reporter assay (2) using LRE, a peak (amplitude) at 120 hours after the stimulation of Dexamezone can be confirmed, but in the reporter assay (1) not using LRE, 60 hours after stimulation. Only the peak (amplitude) at the time point can be confirmed. It is expected that the amplitude and phase of the clock gene expression can be observed over a long period of time using the LRE derived from chromated fireflies.

  From the above results, it is expected that the use of LRE in the existing luminescence reporter assay will show not only the effect of increasing the amount of luminescence but also the effect on the stabilization of phase and amplitude required for clock gene analysis and the like. .

[Example 6: Luminescence imaging analysis of clock gene using LRE derived from chromated fireflies]
In Example 5, a luminescent reporter assay utilizing LRE not only enhances the luminescent signal, but also shows effects on phase and amplitude stabilization. Until now, when imaging a luminescent sample with low luminescence intensity due to low promoter activity, it is difficult to observe individual cells, or it is necessary to lengthen the exposure time to capture a clear image. there were. Thus, the clock gene is analyzed for luminescence imaging using chromatobotanic LRE.

1. Preparation of Expression Vector As in Example 5, mPer2 :: pGL4.12 Vector is prepared.

2. Luminescent promoter measurement using chromatobotan LRE-derived NIH3T3 cells seeded in 35 mm dishes with mPer2 :: pGL4.12 Vector (0.5 μg) and chromatobotan-derived LRE gene pcDNA3.1 (+) vector (2.5 μg) The gene is introduced using the introduction reagent Lipofectamine 2000 (Invitrogen) according to the manual. At 24 hours after gene transfer, clock gene oscillation is synchronized by 100 nM Dexamezone stimulation.

  For luminescence observation, D-luciferin (manufactured by Promega: final concentration 500 μM) is added to the culture solution DMEM-HEPES High Glucose (Gibco 21063-029), and observation is performed using the luminescence imaging system LV200 (Olympus Corporation). . In luminescence observation, exposure is performed for 25 minutes at 30-minute intervals, and the expression level of mPer2 gene over 90 hours is observed. The objective lens is 20 times, the binning is 1 × 1, and the CCD camera is Ixon (manufactured by Andor).

  After the time lapse observation, the captured observation image is stored, and the numerical data is analyzed from the observation image using an image analysis system “MetaMorph (manufactured by Molecular Device Co., Ltd.)” and the analyzed numerical data is graphed.

  An expected view of the acquired image is shown in FIG. (A) shows a bright field image after 30 minutes of observation with LV200, (B) shows a luminescent image after 30 minutes of observation with LV200, and (C) shows a brightfield image and a luminescent image after 30 minutes of observation with LV200. This shows the overlay. Together with cell morphology information, the mPer2 gene promoter activity for each cell, that is, the expression level of mPer2 can be measured.

  9a and 9b are predictions of the results of quantifying the amount of luminescence of cells. As shown in FIG. 9a, seven NIH3T3 cells are selected from the luminescence image. The change with time of the luminescence amount of each cell is shown in FIG. 9b.

  FIG. 9b shows that the amount of luminescence, that is, the expression level of the clock gene mPer2 is different for each cell. Thus, by performing luminescence imaging observation using LRE, it is expected that their behavior can be observed for each cell even when using a promoter with a low expression level such as a clock gene. .

Claims (10)

Imaging a biological sample with a portion containing luciferase, luciferin and luciferin regenerating enzyme over time to obtain a plurality of luminescent images;
An analysis method comprising associating brightness of a region corresponding to each part of each of the plurality of luminescent images with a state of the part or a state of the biological sample.   The method according to claim 1, wherein the biological sample is a cell, an embryo, a tissue, or an individual.   The method according to claim 1, wherein the biological sample further comprises a factor for adjusting the amount of light emitted by the luciferase in the biological sample.   The method according to claim 3, wherein the luciferase is expressed from a luciferase gene introduced into the biological sample, and the factor is a promoter arranged upstream of the luciferase gene.   4. The method according to claim 3, wherein the factor is a cofactor essential for a luminescence reaction catalyzed by the luciferase.   The method of claim 1, wherein the biological sample comprises two or more luciferases that catalyze a distinguishable luminescent reaction.   2. The method of claim 1, wherein the luciferase is fused to a first protein, and the biological sample further comprises a labeled second protein that is different from the first protein.   The first protein is a marker related to a selective proteolysis reaction, a marker consisting of all or part of a protein showing protein interaction, a marker consisting of all or part of a ligand detection protein, a marker of embryo development The method according to claim 7, which is a neuronal differentiation marker or an ES cell differentiation marker.   A luciferin regenerating enzyme protein having the amino acid sequence set forth in SEQ ID NO: 1.   A nucleic acid comprising a luciferin regenerating enzyme gene having the base sequence set forth in SEQ ID NO: 2.