JP2011050258A - Variant red light-emitting luciferase specialized for bioimaging - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a genetic structure which is non-sensitive to pH in cells, emits red light capable of being specified with a filter or the like and having the maximum light wavelength of ≥620 nm, hardly or never damages the cells, and enable imaging in each cell for a long time, and to provide a transformed cell, especially a transformed human cell. <P>SOLUTION: There is provided the variant luciferase obtained by mutating wild type luciferase, emitting red light having wavelengths of ≥620 nm, and having a larger emission intensity than the wild type luciferase. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a gene construct of a mutant red light-emitting luciferase whose expression in a cell is stabilized for the purpose of bioassay and bioimaging of biological information and whose luminescence level is amplified, a combination thereof, The present invention relates to transformed mammalian cells, methods for evaluating gene expression in cells, and cell imaging.

  In the field of life science, it is very important to analyze various phenomena that occur in cells such as fluctuation of intracellular calcium, phosphorylation of intracellular protein, distribution of energy ATP or measurement of gene transcriptional activity. Yes, various molecular probes have been created as means for analysis and imaging has been performed. In particular, various fluorescent proteins are used as intracellular imaging tools. Fluorescent proteins do not require cofactors and have fluorescent activity alone at almost the same time as intracellular expression. Fluorescent protein is used as a monitor protein for protein localization in cells using fluorescence activity as an indicator, but it requires excitation light and is difficult to quantify due to non-uniform fluorescence yield. The cells are damaged by the light and are not suitable for long-term observation.

  Beetle luminescent enzyme luciferase is used as a reporter gene for the evaluation of the influence of foreign factors on cells, propagation of intracellular signal transduction, or expression analysis of individual protein groups. For example, a gene construct in which a transcriptional active region is linked to a firefly luciferase gene is prepared and introduced into a cell. After the cell is treated with a drug for a certain period of time, the cell is collected and lysed, and a luminescent substrate is added. A system for evaluating transcriptional activity by measuring the amount of luciferase synthesized in a cell can be mentioned. Since transcription activity is evaluated from the amount of luminescence of luciferase, it is excellent in quantitativeness, and this system-related product has already been developed and marketed by many companies.

  There are two types of beetle luciferases: a firefly luciferase group that changes the luminescent color in conjunction with pH, and a group of beetle beetles that do not change the luminescent color, and a group of luciferases derived from railroad insects. Among them, in the case of American firefly (Photinus pyralis) luciferase, in vitro, the half-life is about 2-25 times, and the mutant has improved thermostability. It was reported that it is a suitable luciferase (Non-patent Document 1). However, in applications that presume luminescent color discrimination, such as multi-gene expression (multi-gene transcriptional activity measurement system: Katsuhiro Omiya, Yoshihiro Nakajima; Patent Document 1) that focuses on the diversity of luminescent colors, Since luciferase changes the emission color, it cannot be identified as an imaging probe.

  As a beetle luciferase suitable for imaging that does not change the luminescent color, there is a green luminescence luciferase derived from the red beetle. Compared with firefly luciferase, for example, wild-type luciferase derived from American firefly (Photinus pyralis), the intracellular stability is high, and the luminescence intensity is improved 100 times or more (Patent Document 2). When this luciferase is localized in various organelles in a cell, the dynamics of the organelle can be traced over several days through a luminescence signal (Non-patent Document 2). In addition, a mutant luciferase that is genetically modified based on the green luciferase derived from red beetle, which is made for imaging, is produced, and the emission intensity is 1% or less of the wild type, but the maximum emission wavelength is 619 nm ( Patent Document 3).

  As a luciferase that is identifiable by color identification and has strong emission intensity in living cells and is suitable for imaging, and can be distinguished without changing the emission color in conjunction with pH, there are green, orange, and red emission luciferases derived from the red beetle However, the maximum emission wavelength including mutants is only 619 nm or less. The more the maximum emission wavelength is shifted to the longer wavelength side, the easier the color discrimination with the filter and the higher sensitivity and high resolution imaging becomes possible. Moreover, in vivo imaging increases the permeability in the body, enabling highly sensitive measurement.

  It is very important to evaluate the influence of a factor on a living body in evaluating a drug and developing a new drug, or in evaluating the toxicity of a chemical substance. Traditionally, measurement / evaluation using a cell tissue / group from measurement / evaluation using a living organism such as a mouse can evaluate a foreign factor based on changes in information between cells and within a cell. It is happening. Therefore, the application of optical imaging is wide. At this time, luminescence imaging is gaining importance by complementing fluorescence imaging. In particular, in vivo imaging, red luminescence luciferase that is closer to near-infrared light is a powerful light because it does not require external light. Further, when imaging a plurality of cells, it is desirable that the two maximum emission wavelengths are further apart from each other because the color is efficiently divided.

  In order to establish luciferase as an in vivo in vivo, in vitro intracellular and intercellular imaging tool, high stability of luciferase and a relatively long protein life in mammalian cells are desired. Conventional American fireflies, Japanese fireflies, Iriomote fireflies, South American railway insects, and Jamaican red beetles, which have been used for measuring the transcriptional activity of mammalian cells, did not show sufficient luminescence and stability. In particular, American firefly and Japanese firefly luciferase are difficult to measure stably because the emission spectrum changes depending on intracellular pH environment. The only luciferase for imaging whose emission spectrum does not change in a pH environment or the like is only the green luminescence luciferase derived from the red beetle. However, although luciferases include green, orange, and red emission luciferases, none are suitable for imaging in which the maximum emission wavelength exceeds 620 nm or more (Patent Document 2 and Non-Patent Document 2).

WO2004 / 099421 JP 2007-159567 A JP 2008-289475 A JP 2006-55082 A

BaggettB et al .: Thermostability of firefly luciferases affects efficiency of detection by in vivo bioluminescence. Mol Imaging. 2004 Oct; 3 (4): 324-32. Viviani VR (2002) The origin, diversity and structure-function relationships of insect luciferases.Cell Mol Life Sci 59: 1833-1850.

  The present invention is a red light emission having a maximum light emission wavelength of 620 nm or more that is insensitive to intracellular pH and can be distinguished by a filter or the like, and has little or no damage to the cells. It is an object of the present invention to provide genetic constructs and transformed cells, particularly transformed human cells, that enable imaging of the above.

  The present invention also relates to a method for imaging the interaction of each protein fused with each part of the luminescent enzyme by dividing the luminescent enzyme and introducing it into the cell as a fusion protein, an imageable transformed cell, and such a fusion The object is to provide a combination of genetic constructs encoding proteins.

  As a result of various studies to obtain a luciferase derived from a mutant railway worm that emits red light, the present inventors obtained a mutant luciferase gene by performing a mutation introduction treatment on the wild-type luciferase gene, By culturing cells containing the gene construct inserted into the vector DNA, it is possible to obtain a mutant luciferase having a red emission wavelength approximately the same as that of wild-type luciferase and having a strong emission intensity in mammalian cells. The headline and the present invention have been completed.

  That is, the present invention includes the following mutant luciferase, genes encoding the mutant luciferase, gene constructs containing these genes, transformed cells containing the gene constructs, imaging methods using the transformed cells, gene introduction An imaging method using a living organism and a method for measuring transcriptional activity are provided.

Item 1
A red luciferase derived from a railroad worm, wherein the luminescence intensity in mammalian cells is increased by at least 3 times compared to wild-type luciferase.

  Item 2. The mutant luciferase according to Item 1, wherein the emission wavelength of the peak intensity in the mammalian cell is 600 to 635 nm.

  Item 3. The mutant luciferase according to Item 1 or 2, wherein the red luciferase derived from a railroad worm is a genus Phrixothrix.

  Item 4 The mutant luciferase according to Item 3, wherein isoleucine at position 212 and / or asparagine at position 351 is substituted with another amino acid.

  Item 5 The other amino acid to which isoleucine at position 212 is substituted is selected from the group consisting of leucine, asparagine, threonine, serine, methionine, alanine, glycine, valine, tyrosine, cysteine, and phenylalanine. Mutant luciferase.

  Item 6 The mutant luciferase according to Item 5, wherein the other amino acid to which isoleucine at position 212 is substituted is leucine.

  Item 7 The other amino acid in which asparagine at position 351 is substituted is selected from the group consisting of lysine, isoleucine, threonine, serine, methionine, alanine, glycine, valine, tyrosine, cysteine, and phenylalanine. The mutant luciferase according to any one of the above.

  Item 8 The mutant luciferase according to any one of Items 4 to 7, wherein the other amino acid in which the asparagine at position 351 is substituted is lysine.

  Item 9 The mutant luciferase according to Item 1 or 2, encoded by the gene sequence according to any one of SEQ ID NOs: 3 to 7.

  Item 10 A gene encoding the mutant luciferase according to any one of Items 1 to 9.

  Item 11. A vector comprising the gene according to Item 10.

  Item 12 A transformed cell transformed with the gene according to Item 10.

  Item 13 The transformed cell according to Item 12, wherein the cell is a mammalian cell.

  Item 14. The transformed cell according to Item 13, wherein the mammalian cell is a human cell.

  Item 15 A method of using the transformed cell according to any one of Items 12 to 14 for imaging of intracellular organelles.

  Item 16 A method of using the transformed cell according to any one of Items 12 to 14 for measuring transcription activity.

  According to the present invention, it is possible to supply a red luminescent probe suitable for imaging, which retains a luminescent color close to that of a wild-type railway insect-derived red luciferase and has enhanced luminescence intensity in mammalian cells. In this way, intracellular imaging can be multi-colored and multi-colored. In vivo imaging with red light emission, which is highly biopermeable, is also possible.

Comparison of the amount of mutants in cell lysates by Western blotting (A) and comparison of luminescence activity (B) Emission spectra of wild-type and mutant red luciferases Changes in light emission over time in mammalian cells of wild-type and mutant red luciferase (A) and integrated value of light emission over 24 hours (B) Example of in vivo cytoluminescence imaging image of mammalian cells of wild type and mutant red luciferase (SLR-N351K and SLR-I212L / N351K) (A) and integrated value of cell luminescence (B)

  Other terms and concepts in the present invention are defined in detail in the description of the embodiments and examples of the invention. Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known literatures and the like, except for the technique that clearly shows the source. For example, genetic engineering and molecular biology techniques are described in Sambrook and Maniatis, “Molecular Cloning A Laboratory Manual”, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel, FM et al. “Current Protocols in Molecular Biology”, John Wiley. & Sons, New York, N. Y, 2007, etc.

  The mutant luciferase activity of the present invention refers to the activity of an enzyme reaction using luciferin as a substrate, and is measured by detecting light emitted when the substrate luciferin returns to the ground state after being excited by the enzyme reaction. Is done.

  The railroad worm-derived red luminescent luciferase in the present invention is a red luminescent luciferase derived from a railroad worm, and a specific example thereof includes the amino acid sequence shown in SEQ ID NO: 1. An example of a gene sequence encoding this is the polynucleotide sequence shown in SEQ ID NO: 2.

  The red light emitting luciferase in the present invention is an enzyme that can emit light having a maximum wavelength of 600 to 635 nm in the presence of luciferin. More preferably, the maximum wavelength is 620 to 635 nm.

  Examples of the mammal of the present invention include humans, cows, horses, sheep, monkeys, pigs, mice, rats, hamsters, guinea pigs, rabbits and dogs. Preferred are human, monkey, mouse, rat and hamster. Most preferred is a mouse.

  The mammalian cell in the present invention may be a primary cultured cell obtained from the above mammal or a cell line that has been immortalized. Examples of cell lines include human-derived Hela cells, African green monkey-derived COS7 cells, mouse-derived 3T3 cells, hamster-derived CHO cells, rat-derived PC12 cells, and the like.

  The mutant luciferase of the present invention can emit a maximum emission wavelength in the presence of approximately the same level of luciferin as compared to wild-type luciferase. The difference in the maximum emission wavelength is in the range of 20 nm or less. More preferably, it is 15 nm or less, More preferably, it is 10 nm or less. That is, the range may be any range that does not deviate from the red range in which the wild-type luciferase can emit light in the presence of luciferin.

  The isoleucine at position 212 in the present invention is included in the above-mentioned gene sequence of the worm-derived luciferase in a site that hybridizes with the gene sequence described in SEQ ID NO: 8 under stringent conditions. It shows that the gene corresponding to cTC (bold part) in the gene sequence is an encoded amino acid. A specific example is isoleucine at position 212 in the amino acid sequence set forth in SEQ ID NO: 1.

  In the introduction of mutation into isoleucine at position 212 in the present invention, the amino acid to be substituted may be selected so as to achieve the purpose of increasing the luminescence intensity of the red luminescent luciferase in mammalian cells. Specific examples of amino acids in which isoleucine at position 212 is substituted include leucine, asparagine, threonine, serine, methionine, alanine, glycine, valine, tyrosine, cysteine, and phenylalanine. Preferably, valine or leucine can be selected for the purpose of adjusting hydrophobicity. More preferred is leucine.

  The asparagine at position 351 in the present invention is included in a site that hybridizes under stringent conditions with the gene sequence described in SEQ ID NO: 10 in the gene sequence of the above-mentioned rail worm-derived luciferase. It shows that it is an amino acid encoded by the gene corresponding to AAg (bold part) in the base sequence of. A specific example is asparagine at position 351 in the amino acid sequence set forth in SEQ ID NO: 1.

  In the present invention, mutation can be introduced into lysine at position 351 by selecting a substituted amino acid so as to achieve the purpose of increasing the luminescence intensity of red luminescent luciferase in mammalian cells. Specifically, lysine, isoleucine, threonine, serine, methionine, alanine, glycine, valine, tyrosine, cysteine, phenylalanine and the like are listed as amino acids in which asparagine at position 351 is substituted. Preferred is arginine, lysine having a positive charge. More preferred is lysine.

  The mutant luciferase of the present invention is a railroad worm-derived red luciferase, which is a mutant luciferase having an increased luminescence intensity when expressed in mammalian cells compared to the wild type. Examples of the gene for the red luciferase derived from a railroad worm include any one of SEQ ID NOs: 3 to 7. Accordingly, a mutant in which isoleucine at position 212 and / or asparagine at position 351 in the wild-type luciferase defined by the gene of SEQ ID NO: 1 is substituted with another amino acid residue is exemplified.

The stringent condition in the present invention refers to a condition in which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions are in “1 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0,2 M NaH ₂ PO ₄ , 20 mM EDTA · 2Na, pH 7.4). The condition of “hybridizing at 42 ° C. and further washing with 0.5 × SSC at 42 ° C.” is one example of the stringent conditions of the present invention, but is not limited thereto.

  The vector containing the mutant luciferase gene of the present invention includes a multiple cloning site for introducing the mutant luciferase gene, a promoter sequence, an enhancer sequence, a polyadenylation sequence, an insulator sequence, a selectable marker gene sequence, and a replication as necessary. It may have a starting point. Here, when expressing the mutant luciferase of the present invention as a recombinant using a mammalian cell as a host cell, it may further have a Kozak sequence as a transcription initiation site and a strong promoter such as a CMV promoter. desirable.

  The method for introducing a vector containing the mutant luciferase gene of the present invention into a mammalian cell can be carried out by selecting a known method suitable for the use after cell introduction. Specific methods include lipofection method, particle gun method, electroporation method, calcium phosphate method and the like.

  The luminescence intensity in the mammalian cells in the present invention is determined by culturing the transformed cells into which the mutant luciferase gene has been introduced by the above-described method in an appropriate medium containing luciferin as a substrate for the mutant luciferase. After luciferin is taken into the transformed cells, the luminescence obtained by the enzymatic reaction occurring in the transformed cells can be measured. A known method can be used for the measurement, and the measurement can be performed by a known method using a luminometer by a photon counting method or the like.

  Furthermore, by using a microscope equipped with a photon counting device, it is possible to image a cultured cell level, a cell unit of a cultured cell, or an organelle in a cell. Therefore, it is possible to perform long-time real-time measurement and in vivo imaging in which cells are made alive by providing an incubator function in the observation atmosphere of the microscope.

  By using the mutant red luminescence luciferase gene of the present invention, it can be used for the uses described in Patent Document 2. Specific examples are shown below.

    When a mammalian cell is transformed with the mutant gene construct of the present invention, the transformed cell can obtain a sufficiently high amount of luminescence. When a heterogeneous protein or tag is bound, the amount of luminescence is reduced, so even if nothing is bound, it is difficult to bind the heterologous protein or tag with a conventional gene construct that does not emit enough light. On the other hand, in the present invention, since the amount of light emitted by a luminescent enzyme that binds a heterologous protein or tag is very high, imaging of individual mammalian cells can be performed in a state where the heterologous protein or tag is bound.

  The heterologous protein fused with the luminescent enzyme of the present invention includes any heterologous protein, and the tag fused with the luminescent enzyme of the present invention includes a PEST sequence or ubiquitin, or a biologically active fragment thereof, or these. Protein destabilization signal, nuclear localization signal, membrane localization signal, cytoplasmic localization signal, mitochondrial localization signal, ER localization signal encoded by a nucleotide sequence encoding a variant or derivative of Can be mentioned. By using the heterologous protein or tag fused with the luminescent enzyme of the present invention as described above simultaneously with another heterologous protein or tag fused with imaging luciferase having a different luminescent color, the organelle in the cell is luminescent. Can be identified by the difference in color.

    The destabilizing factor may use a PEST sequence or the like that destabilizes the luminescent enzyme protein. The destabilizing factor lacks poly A signal, c-fos, c-jun, c-myc, GM-CSF, The sequences derived from various genes such as IL-3, TNF-α, IL-2, IL-6, IL-8, IL-10, urokinase, bcl-2, Cox-2, and PAI-2 are used as luminescent enzyme genes. The mRNA of the luminescent enzyme may be destabilized by ligation.

    By destabilizing the luminescent enzyme protein or its mRNA, changes in the expression level of the luminescent enzyme in response to a certain stimulus can be observed more accurately (without time lag). This is realized for the first time by the gene construct of the present invention whose expression efficiency in mammalian cells is higher than that of fireflies.

    The PEST sequence used as a tag is preferably the 3 ′ end of ornithine decarboxylase or a variant thereof, and the 3 ′ end of ornithine decarboxylase or a variant thereof is preferably derived from a mammal, and is commonly used. Is derived from a mouse. PEST refers to an amino acid sequence rich in proline (P), glutamic acid (E), serine (S), and threonine (T), and it is known that a protein containing the PEST sequence has a short half-life.

  In one embodiment of the present invention, a luciferase split assay, wherein the luminescent enzyme is divided into, for example, an N-terminal portion and a C-terminal portion, and the DNA encoding each is linked to a heterologous protein, and these gene constructs Are co-expressed in one cell. Here, the N-terminal part of the luminescent enzyme and the C-terminal part of the luminescent enzyme are expressed separately, but they can be designed to emit light when they are in close proximity.

  The activity of the mutant luciferase of the present invention can also be measured in a state contained in a cell lysate of a mammalian cell of the host. The host cell can be lysed using a known method. Specifically, the host cells are lysed by homogenization or ultrasonic disruption with a physiological buffer containing a surfactant. The buffer solution may contain a protease inhibitor. By using a cell lysate containing a mutant luciferase obtained by such a method, the activity of luciferase can be measured.

  As the luciferin used as the substrate of the mutant luciferase in the present invention, a known luciferin can be used. Specifically, D-firefly luciferin, L-firefly luciferin, D-firefly luciferin 6 'benzyl ether (luciferin-6'-benzyl ether, D-firefly luciferin 6' methyl ether (luciferin-6'-methyl ether, etc.) It is done.

   The mutant luciferase activity of the present invention can be measured by a known method using a luminometer such as a photon counting method after mixing with a cell lysate of a host cell containing the mutant luciferase. Similarly, the emission spectrum can be measured by a known method. Here, luciferin serving as a substrate is present in a form contained in a physiological buffer used for lysis of host cells, and may be reacted with mutant luciferase simultaneously with the cell lysis operation.

  The method for producing a mutant luciferase in the present invention can be obtained by synthesizing in a cell and then purifying it. As a specific example, it can be carried out using a method similar to the method for producing wild-type luciferase described in Patent Document 4.

  A known method can be referred to for a method for measuring transcriptional activity using a transformed cell into which the mutant luciferase gene of the present invention has been introduced. A specific example is described in Patent Document 2. A method is mentioned.

  Specifically, a gene cassette having a transcriptional activity (promoter activity) upstream of the mutant luciferase gene in the present invention, or a test sequence that may possibly have been introduced, is introduced into cells, or by homologous recombination or the like. A transformed cell in which a gene having a mutant luciferase gene is incorporated under a certain test sequence is prepared. Thereafter, the transcriptional activity of the test control sequence can be measured using the activity of the mutant luciferase expressed under the transcriptional control of the test sequence as an index. The method for introduction into cells and the method for measuring the activity of mutant luciferase can be carried out by the methods described above.

   It should be noted that a person skilled in the art can easily modify the mutant luciferase gene of the present invention so long as it does not diminish the expectation of each function described above, such as mutation, substitution, deletion, insertion, and tandemization. It can be implemented.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, it cannot be overemphasized that this invention is not limited to these Examples.
Example 1
Based on a vector that contains the wild-type amino acid sequence of red light-emitting luciferase (Phrixothrix RED: PhRED) derived from a railroad worm and a cDNA (SEQ ID NO: 2) in which the codon usage has been changed to a mammalian type inserted downstream of the pCMV promoter. did. Using this plasmid as a template, site-specific mutation sites were introduced by the inverse PCR method so that the three amino acid residues of luciferase could be changed to other residues. Mutagenesis is carried out using the site-specific mutagenesis kit KOD-Plus-Mutagenesis Kit (Toyobo) according to the instruction manual supplied with the kit, and the resulting plasmid is used to characterize Escherichia coli competent cell DH5α (Toyobo). Made a conversion. That is, the position of the codon corresponding to the 212th Ile was set to CTC (the bold site in Table 1), and this was designated as Primer # 1 (SEQ ID NO: 8). Also, the complementary strand in the reverse direction of back-to-back was designated as Primer # 2 (SEQ ID NO: 9). As a result, a mutant SLR-I212L in which the 212th Ile was changed to Lue was constructed. The position of the codon corresponding to the 351st Asn was set to AAG (the bold portion in Table 1), and this was designated as Primer # 3 (SEQ ID NO: 10). Also, the complementary strand in the reverse direction of back to back was designated as Primer # 4 (SEQ ID NO: 11). Thus, a mutant SLR-N351K in which the 351st Asn was changed to Lys was constructed. The position of the codon corresponding to the 463rd Ser was set as CGT (the bold portion in Table 1), and this was designated as Primer # 5 (SEQ ID NO: 12). Also, the complementary strand in the reverse direction of back to back was designated as Primer # 6 (SEQ ID NO: 13). As a result, the 463rd Ser constructed SLR-S463R which was changed to Arg. The sequence of the mutation was confirmed with a sequencer (ABI3100; Applied Biosystems). Based on site-specific mutants in which one amino acid residue was mutated, mutations were further added, and finally 5 types (SLR-I212L; SEQ ID NO: 3, SLR-N351K; SEQ ID NO: 4, SLR-S463R; SEQ ID NO: 5, SLR-I212L / N351K; SEQ ID NO: 6, SLR-I212L / S463R; SEQ ID NO: 7).

Example 2
The created vector into which the mutation was introduced was introduced into NIH3T3 cells derived from mice. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) 10% FBS medium at 37 ° C. with 5% CO ₂ . The day before gene introduction, the number of cells per 35 mm dish was 3 × 10 ⁵ cells. The gene was introduced using Lipofectamine PLUS (Invitrogen) with a gene plasmid amount of 1000 ng / dish. 48 hours after gene introduction, the cells were collected, washed with PBS, dissolved in 150 μl of Takara Bio's cell lysing agent (M-PER Mammalian Protein Extraction Reagent), and luciferin solution ((( 50 μl of LARII (Promega) was added, and the luminescence activity was immediately measured by integrating for 20 seconds using ATTO (AB-2250), while the amount of protein in the cell lysate was measured using the BioRad Protein Assay kit. 2 μg of the sample was subjected to SDS-PAGE electrophoresis, transferred to a membrane and subjected to Western blotting.An antiserum prepared by immunizing a rabbit with purified red luciferase as a primary antibody was diluted 2500 times and used. As secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG (Jackson ImmunoResearch) was used, and luminescence was generated by ECL Western Blotting Detection Reagents (GE Healthcare Bioscience). The light emission was imaged with LAS-4000miniPR (Fuji Film) (I think that the description is not enough and I added a little, so please check it). The protein expression was the same, although there was a slight difference.Figure 1B shows the individual luminescence levels normalized by the protein expression level, which indicates that SLR-I212L, The amount of luminescence increased by about 8 times with three types of SLR-I212L / N351K and SLR-I212L / S463R.

Example 3
The emission spectra of various mutants were measured. AB-1850S manufactured by Atto Corporation was used, and 15 μl of luciferin solution ((LARII; Promega) was added to 15 μl of cell lysate. Among 5 mutants, SLR-I212L, SLR-S463R, SLR-I212L / S463R was almost the same as the wild type, but SLR-N351K and SLR-I212L / N351K blue-shifted the entire emission spectrum by about 10 nm, but the maximum emission wavelength of all mutants was 620 nm or more (Fig. 2).

Example 4
Genes were introduced into NIH3T3 cells derived from mice in which 1000 ng / dish of various plasmids prepared in Example 1 were cultured in 35 mm dishes. Twenty hours after gene introduction, 100 μM D-luciferin (10% FBS in DMEM medium, 20 mM Hepes / NaOH (pH 7.0)) was replaced, and placed in a continuous luminescence measuring device Ato AB-2500, 5% CO ₂ 20 In an environment of 0 ° C., the change in luminescence activity was measured over about 30 hours while integrating the amount of luminescence for 1 minute at 20 minute intervals. FIG. 3A shows the change in the luminescence activity of the wild type and various mutants, and repeated to confirm the reproducibility. As a result, it has been clarified that SLR-N351K and SLR-I212L / N351K show high light emission. In order to quantitatively examine this difference, the amount of luminescence for 24 hours was integrated. FIG. 3B shows the integrated amount, and both SLR-N351K and SLR-I212L / N351K showed that the amount of luminescence was improved 5 times or more compared to the wild type.

Example 5
Since the luminescence intensity was improved in the mutants in the living cells, in vivo cell imaging of SLR-N351K and SLR-I212L / N351K with increased wild type and luminescence values was performed. After introducing the same amount of gene vector into mouse-derived NIH3T3 cells, luminescence was measured with Cellgraph AB-3000B manufactured by Atto Corporation. FIG. 4A is an example of an in vivo cytoluminescence imaging image, and repeated images were collected and confirmed. Images with particularly high emission intensity were obtained with SLR-N351K. FIG. 4B shows the result of analysis using the software of Metamorph (Universal Imaging, Brandywine) in order to clarify the change in the light emission amount. It became clear that SLR-I212L / N351K, which is 4 times or more of SLR-N351K compared to the wild type, can obtain a bright image of 3.5 times or more.

  As a result, wild-type red luciferase is the luciferase containing the most red wavelength. It became clear that the / N351K mutant was practical.

  The present invention provides a mutant luciferase that exhibits high intensity luminescence in living cells having a peak wavelength of 620 nm or more as a beetle luciferase. In particular, it is possible to obtain a more stable luciferase derived from a mutant red railway worm with a higher intensity of luminescence than a wild-type railway worm-derived luciferase gene. These mutant red railworm-derived luciferases are suitable for in vivo luminescence imaging because of their excellent cell or tissue permeability. In addition, in the ATP detection, it is possible to improve the measurement sensitivity when measuring the substance in a colored solution such as red, for example, the ATP content in blood. Furthermore, it is possible to use two color reporter genes using two types of mutant luciferases with wild type and mutant type, or different peak emission wavelengths. For example, the transcriptional activity of multiple genes can be measured simultaneously. It can be used for a wide variety of industries such as drug discovery, medical care, and food inspection.

Claims (16)

A red luciferase derived from a railroad worm, wherein the luminescence intensity in mammalian cells is increased by at least 3 times compared to wild-type luciferase. The mutant luciferase according to claim 1, wherein the emission wavelength of the peak intensity in the mammalian cell is 600 to 635 nm. The mutant luciferase according to claim 1 or 2, wherein the red luciferase derived from a railworm is a genus Phrixothrix. The mutant luciferase according to claim 3, wherein isoleucine at position 212 and / or asparagine at position 351 is substituted with another amino acid. The mutation according to claim 4, wherein the other amino acid substituted with isoleucine at position 212 is selected from the group consisting of leucine, asparagine, threonine, serine, methionine, alanine, glycine, valine, tyrosine, cysteine, and phenylalanine. Type luciferase. 6. The mutant luciferase according to claim 5, wherein the other amino acid to which isoleucine at position 212 is substituted is leucine. The other amino acid in which asparagine at position 351 is substituted is selected from the group consisting of lysine, isoleucine, threonine, serine, methionine, alanine, glycine, valine, tyrosine, cysteine, and phenylalanine. The mutant luciferase according to any one of the above. The mutant luciferase according to any one of claims 4 to 7, wherein the other amino acid in which asparagine at position 351 is substituted is lysine. The mutant luciferase according to claim 1 or 2, which is encoded by the gene sequence according to any one of SEQ ID NOs: 3 to 7. A gene encoding the mutant luciferase according to any one of claims 1 to 9. A vector comprising the gene according to claim 10. A transformed cell transformed with the gene according to claim 10. The transformed cell according to claim 12, wherein the cell is a mammalian cell. The transformed cell according to claim 13, wherein the cell is a human cell. 15. A method of using transformed cells according to any one of claims 12 to 14 for imaging of intracellular organelles. The method using the transformed cell in any one of Claims 12-14 for a transcriptional activity measurement.

Images