WO2011102178A1

WO2011102178A1 - Analysis system using modified reporter gene

Info

Publication number: WO2011102178A1
Application number: PCT/JP2011/050957
Authority: WO
Inventors: 誠培金; 博明田尾
Original assignee: 独立行政法人産業技術総合研究所
Priority date: 2010-02-19
Filing date: 2011-01-20
Publication date: 2011-08-25

Abstract

Disclosed is a reporter gene which, in an analysis method using a reporter gene such as the reporter gene assay method or the two-hybrid assay method, makes it possible to shorten the external stimulus time to the cell, and can make it possible for the intensity of luminescence caused by the reporter gene expression in response to the external stimulus to quantitatively and accurately reflect said degree of external stimulus. In the analysis method for analyzing the amount or timing of target gene expression in a cell in response to an external stimulus, at least one of the amino acid residues at positions corresponding to positions 89 to 118 in the amino acid sequence of Gaussia Luciferase (GLuc) is replaced in the amino acid sequence of a luminescent enzyme of a marine animal as a reporter gene, said replacement being a conservative　amino　acid　replacement of the amino acid residue(s) in at least one position chosen from the positions corresponding to positions 89, 90, 95, 97, 100, 108, 112, 115 and 118 in said amino acid sequence, and a gene is used encoding the altered luminescent enzyme with thusly improved luminescence.

Description

Analysis system using improved reporter gene

The present invention uses a reporter gene, characterized in that a marine animal luminescent enzyme variant gene having improved luminescence intensity, stability, and / or animal tissue permeability by long wavelength shift is used as a reporter gene. The present invention relates to an improvement method for various analysis systems.

Measurement of molecular phenomena in vivo (for example, protein structural changes, phosphorylation, protein-protein interactions, production of secondary signaling substances, etc.) understand life phenomena and elucidate the molecular mechanisms involved It is extremely important above. Conventionally, various methods have been developed as a method for analyzing such a molecular phenomenon. Main methods are (a) fluorescent resonance energy transfer (FRET; Non-Patent Documents 1 and 2), (b) bioluminescent resonance energy. transfer (BRET; Non-Patent Document 3), (c) Protein complementation (Non-Patent Documents 4 and 5), (d) Measurement of transcriptional activity based on the binding affinity between a chemical substance and a receptor, etc. Examples include reporter gene assay (non-patent document 6) and (e) a two-hybrid assay (non-patent document 7) for analyzing protein interactions.
Reporter gene assays and two-hybrid assays, in particular using reporter genes, are the most universal and standard bioanalytical methods today, and the types of reporter genes used here are fluorescent protein genes (hereinafter referred to as fluorescent methods). The photoprotein gene (hereinafter referred to as the luminescence method) is mainly used. The fluorescent method is characterized by expressing a green protein (GFP) or a variant thereof as a reporter gene product. However, the fluorescent protein has a high background due to autofluorescence and an external light source. And requires a large-scale device such as a fluorescence microscope and a precise filter system. There is also a problem that it takes several hours to several days at the shortest time for the fluorescent chromophore to mature. In addition, when using a fluorescence microscope, there is a limit to the number of cells that can be observed at one time, and there is a problem in quantitativeness (Non-patent Document 8).

On the other hand, the luminescent method is characterized by expressing a bioluminescent enzyme (luciferase) when detecting an external stimulus. Compared with the fluorescence method, the luminescence method does not require a large-sized device and is used in many assay methods using many reporter genes today because of the ease of measurement. Despite its strengths, it also includes issues to be solved. The first reason is that the bioluminescent enzyme is generally lower in luminance than the fluorescent dye. Since the luminance is lower than that of fluorescent dyes, the luminescence method requires a highly sensitive measuring device and is not suitable for single-cell imaging or molecular phenomenon search at the organelle level. In addition, in order to obtain sufficient luminance, it is necessary to take a long stimulation time for the expression product to accumulate. The second reason is that many of the bioluminescent enzymes can only provide monotonous color development and there are few color choices. Advantages of multicolor emission include (a) simultaneous measurement of multiple signals and (b) good biological tissue permeability of long wavelength emission.
Meanwhile, chloramphenicol acetyltransferase (CAT), β-galactosidase (β-gal), alkaline phosphatase (AP) and the like have been widely used as reporter genes. However, the sensitivity of the CAT standard method is generally 10 to 1000 times inferior to that of the luminescent enzyme method (Non-patent Document 9). Similarly, β-galactosidase and alkaline phosphatase are also insensitive, so a long stimulus is required until the expression of these proteins is fully accumulated, and a reaction time of 15 minutes to 1 hour is also required for the color reaction. And
Thus, a common big problem of the conventional assay method using a reporter gene is that a long ligand stimulation time is required until measurement. For example, a standard method requires a stimulation time of about one day. Because it is necessary, it is impossible to measure dynamic molecular phenomena that occur in cells. In addition, in the standard protocol of the assay method using a conventional reporter gene, since the measurement is started after the cells are ground (making a lyset), it takes about 10-20 minutes to make the cell lyset. It was a cause of slowness.
Therefore, there is a demand for shortening the ligand stimulation time in the conventional assay method using a reporter gene, particularly a reporter gene assay and a two-hybrid assay equipped with a luminescent enzyme (luciferase). Development and application of luciferase variants that exhibit bioluminescence and / or bioluminescence shifted to longer wavelengths have been desired.

An object of the present invention is to provide an improved method for shortening the ligand stimulation time in various assay methods using a reporter gene such as a reporter gene assay or a two-hybrid assay. It is an object of the present invention to provide a method for using a genetically modified product that expresses a luciferase that exhibits a stable and bioluminescent light emission and / or bioluminescence shifted to a longer wavelength side.

The present inventors have already introduced a mutation at a specific position of a luminescent enzyme derived from marine animals such as Gaussia luciferase (GLuc), so that luminescence intensity is high and stable, and luminescence shifted to a long wavelength is emitted. It has been clarified that it is possible to synthesize an ultra-bright bioluminescent enzyme efficiently in mammalian cells by codon optimization of the modified gene (specialty). (Application 2009-101025).
The reporter gene is recognized as a reporter gene based on the recognition that the most direct cause is the low brightness of the reporter protein itself. It came to mind using the said mutant luminescent enzyme modified gene which the inventors developed.
Specifically, a reporter gene assay system was constructed by adopting a high-luminance bioluminescent enzyme gene as a reporter gene mounted downstream of a reporter expression plasmid having an androgen response element (ARE) (Example) 1) However, compared with the case of the plasmid (pARE-GLuc) loaded with the conventional GLuc itself, it showed stronger bioluminescence and was able to detect with high sensitivity. Due to the strong bioluminescence, even a small amount of reporter protein can be detected with the existing detector, and the waiting time until the reporter protein accumulates can be reduced. In the two-hybrid assay system, the same results were obtained in less than half of the time required for the two-hybrid assay system. The assay time was shortened (Example 2). In addition, in order to investigate the optimum measurement conditions of the assay system using these reporter genes, examination of the optimum mixing ratio of each plasmid constituting the system (Example 3), the optimum stimulus and its appropriate stimulus concentration (Example) 6) was also performed.
Furthermore, the present invention is a bioanalytical method using kinetics of luminescence intensity by the expressed ultra-bright variant luminescent enzyme, wherein the change in luminescence value per unit time (RLU / sec) is an analysis index. As a result, it was found that quantitative ligand measurement was possible (Example 4), and female hormone selectivity and response sensitivity were also measured (Examples 5 and 6).
Thus, the present invention relating to an analysis system using a marine animal-derived luminescent enzyme variant gene as a reporter gene was completed.

That is, the present invention is specifically as follows.
[1] A reporter gene used in an analysis method for analyzing the timing or amount of expression of a target gene in a cell in response to an external stimulus, and in the amino acid sequence of a marine animal luminescent enzyme, Gaussia luciferase (GLuc) At least one of the amino acid residues at positions corresponding to positions 89 to 118 in the amino acid sequence is substituted, and the substitution is performed at

positions

89, 90, 95, 97, 100, Conservative amino acid replacement for amino acid residues at positions selected from at least one of positions corresponding to positions 108, 112, 115, and 118, thereby improving luminescence function A reporter gene comprising a gene encoding the mutated luminescent enzyme.
[2] The gene encoding the mutant luminescent enzyme is located at

position

89, 90, 97, 108, 112, 115, or 118 on the amino acid sequence of GLuc in the amino acid sequence of marine animal luminescent enzyme. Substitution from the hydrophobic amino acid residue at the corresponding position to another hydrophobic amino acid residue, or from the hydrophilic amino acid residue forming a hydrogen bond at the 95th or 100th position to another hydrophilic amino acid residue The reporter gene according to the above [1], which encodes a mutant luminescent enzyme having a conservative amino acid substitution including a substitution for.
[3] The amino acid residue at the position corresponding to the 90th, 108th, 112th, 115th or 118th position on the amino acid sequence of GLuc in the amino acid sequence of marine animal luminescent enzyme, wherein the conservative amino acid substitution is Substitution with leucine (L), tryptophan (W) or valine (V), substitution of amino acid residue corresponding to position 89 or 97 with tryptophan (W), or equivalent to position 95 The reporter gene according to [2], which comprises substituting the amino acid residue at the position corresponding to glutamic acid (E) or the amino acid residue corresponding to position 100 with asparagine (N) .
[4] A conservative amino acid substitution, wherein the gene encoding the mutant luminescent enzyme comprises substituting leucine for the amino acid residue at position 90 on the amino acid sequence of GLuc in the amino acid sequence of the marine animal luminescent enzyme The reporter gene according to any one of the above [1] to [3], which is a mutant luminescent enzyme gene to which is applied.
[5] Any of the above [1] to [4], wherein the marine animal luminescent enzyme is any one of luminescent enzymes selected from Gaussia luciferase (GLuc) and Kaishi luciferase (MLuc, MpLuc1, and MpLuc2) A reporter gene according to any one of the above.
[6] An expression vector comprising the reporter gene according to any one of [1] to [5] downstream of a response sequence to an external stimulus or a response sequence to a fusion protein formed by a response to the external stimulus.
[7] A transformed cell into which the expression vector according to [6] is introduced.
[8] An analytical method for analyzing the timing or expression level of a target gene in a cell in response to an external stimulus, wherein the transformed cell according to [7] is used.
[9] The intensity of the external stimulus is quantified using the rate of increase in bioluminescence intensity (RLU / sec) based on the expression of the reporter gene after addition of the substrate or the area of the response curve as an index. The analysis method according to [8] above.
[10] The analysis method according to [8] or [9], wherein the analysis method is a reporter gene assay method or a two-hybrid assay method.
[11] Analysis characterized in that, among the ratios of the three types of plasmids constituting the two-hybrid assay method according to [10], the ratio of the plasmid for reporter expression is increased more than twice that of other plasmids. Law.

The present invention, in an analysis system using a reporter gene such as a conventional reporter gene assay method or a two-hybrid method, uses a marine animal-derived luminescent enzyme high-intensity variant gene developed by the present inventors as a reporter gene. The assay time could be shortened. Moreover, in each assay system, it was possible to provide an excellent improved method with high sensitivity and simple quantitative measurement. This is because the reporter gene assay has been applied to the comprehensive detection methods of endocrine disruptors, the diagnosis of hormonal abnormalities in vivo, and the two-hybrid method has been applied to improve the accuracy of proteome analysis. Not only can this be achieved, it can also be expected to be a tool for observing molecular phenomena at the intracellular or organelle level, which has been impossible in the past, in a short time.

Hydrophobic distribution map of amino acid sequences of luminescent enzymes derived from various marine animals. Table of amino acid sequences of luminescent enzymes derived from various marine animals. The dotted box is the putative enzyme active site. Mutation introduction location map in the amino acid sequence of GLuc. Comparison diagram of bioluminescence intensity of each GLuc mutant (1). It is the graph which compared the peak value of the light emission spectrum, and has shown the relative bioluminescence intensity compared with the light emission intensity (left end) of original GLuc. Here, since the cell lysis time is 5 minutes, the organelle is not lysed. Comparison diagram of bioluminescence intensity of each GLuc mutant (2). It is the graph which used the area of each luminescence spectrum measured with the luminescence plate reader as a parameter | index, and has shown the relative bioluminescence intensity compared with the luminescence intensity (left end) of original GLuc. Here, since the cell lysis time was set to 20 minutes, a uniform cell lysate dissolved up to the organelle was obtained. For some high-luminance GLuc mutants, the maximum emission wavelength is also shown. Red shift of the bioluminescence spectrum of GLuc mutant. From above, when I90 is mutated to Tyr (Y), H95 and D100 are mutated to Q and N, and H97 is mutated to W, respectively. Red shift of the bioluminescence spectrum of GLuc mutant. Emission spectra of Y97W, I90L, F89W / I90L / H95E / Y97W (Mon3) compared to conventional GLuc. Relative intensity of long wavelength bioluminescence intensity using 610nm Long-Pass filter. Wavelength of each luminescent variant compared to conventional GLuc (white bar). It can be seen that the mutant exhibits strong long wavelength bioluminescence. Comparison of bioluminescence spectra of representative luciferases. The GLuc mutant (black line) shows much stronger bioluminescence intensity than conventional GLuc (dotted line), FLuc and click beetle luciferase (CBLuc). Comparison of bioluminescence intensity over time. (A) The result of observing the change in bioluminescence over time for a short time (5 seconds) after introducing a substrate (dissolved in PBS buffer) into each mutant. It can be seen that I90L, H95E, and Y97W exhibit relatively stable bioluminescence. (B) The result of observing the change in bioluminescence over time for a relatively long time (10 minutes) after introducing a substrate (dissolved in PBS buffer) into each mutant. While the original GLuc luminescence value (white circles) decreased dramatically, the luminescence values of I90L (black squares) and I90V (gray triangles) showed relatively stable luminescence intensity. Comparison of bioluminescence intensity over time. (C) The result of observing the change in bioluminescence over time for a short time (5 seconds) after introducing a substrate (dissolved in Matthew modified buffer) into each mutant. (D) The result of observing the change in bioluminescence over time for each second after introducing a substrate (dissolved in Matthew modified buffer) into each mutant stepwise. It can be seen that each mutant is very stable and exhibits strong bioluminescence. Luminescent properties of MpLuc1 (Metridia pacifica-derived luciferase) mutant. (A) Emission spectra of MpLuc1 itself, MpLuc1 I114L mutant (I114L), and MpLuc1 Y113W / I114L / H119E / Y121W mutant (MpLuc4). (B) Measurement of long wavelength bioluminescence value (610 nm or more) of MpLuc1 mutant using 610 nm Long-Pass filter. Measurement of luminescence stability of MpLuc1 mutant. Both I114L and MpLuc4 show stable bioluminescence compared to the conventional MpLuc1 itself. Luminescent properties of MLuc (Metridia longa-derived luciferase) mutant. (A) Luminescence spectra of MLuc itself, MLuc I123L mutant (I123L), MLuc Y122W / I123L / H128E / Y130W mutation (MLuc4). (B) Measurement of luminescence stability of MLuc mutant. MLuc4 shows more stable bioluminescence than conventional MLuc itself. Luminescent properties of MLuc mutant. (C) Measurement of long wavelength bioluminescence value (more than 610nm) of MLuc mutant using 610nm Long-Pass filter. (D) Temporal change in luminescence value of MLuc mutant before and after substrate introduction. Example (1) of modifying the nucleotide sequence of a GLuc variant suitable for expression in mammals including humans. The base sequence of the I90L mutant of GLuc, which consists of codons suitable for mammals including humans and introduced with a restriction enzyme site. The underline indicates the position of the restriction enzyme site, and the italic letters indicate the mutation site. Example (2) of modification of the base sequence of a GLuc variant suitable for expression in mammals including humans. Nucleotide sequence of a GLuc enzyme mutant (Mon3 with mutations at four positions including I90L) that consists of codons suitable for mammals and has EcoRV restriction enzyme sites introduced. The underline indicates the position of the restriction enzyme site, and the italic letters indicate the mutation site. A reporter gene assay method using the luminescent enzyme variant of the present invention as a reporter gene. (A) Operation principle diagram of reporter gene assay method. A transcription factor is activated in the presence of a ligand, leading to expression of a reporter protein. (B) Comparison of luminescence intensity in the reporter gene assay when the conventional Gaussia luciferase (GLuc) is mounted and when the luminescent enzyme mutant of the present invention is mounted. It can be confirmed that the area of the luminescence intensity graph is wider when the mutant luminescent enzyme is used. (C) The comparison graph of the light emission area obtained by said (B). A two-hybrid assay method using the reporter gene of the present invention. (A) Operation principle diagram of the two-hybrid assay method. ER LBD Y537 is phosphorylated in the presence of a ligand. This phosphorylation is recognized by the adjacent Src SH2 domain and binding between the fusion proteins occurs. The restoration of the Gal4 transcriptional activity leads to the expression of the reporter protein. (B) Transition of relative light emission intensity by each stimulation time. As a result of applying the ligand stimulation for 6 hours, the case where the high-intensity mutant of the present inventor was mounted showed more superior light emission intensity and S / N ratio compared to the case where the conventional GLuc was mounted. A two-hybrid assay method using the reporter gene of the present invention. (C) Change in bioluminescence value with time when the original GLuc was installed (pG5-GLuc). (D) Change in bioluminescence value with time when the GLuc mutant of the present invention is mounted (pG5-I90L). The absolute value of luminescence is higher than that of GLuc, and the bioluminescence is efficiently increased by a short female hormone stimulation. A two-hybrid assay method using the reporter gene of the present invention. Bioluminescence values (measured with a luminescence photometer) after female hormone stimulation when GLuc and GLuc mutant (9096) are loaded on pG5 vector (0 hours, 3 hours, 6 hours, 18 hours). In the conventional method, the activity of the ligand could not be separated by the stimulation for 3 hours, but in this method, a significant difference in luminescence intensity could be observed even in 3 hours. In the figure, “9096” indicates a GLuc mutant (I90L / T96S). Correlation between the amount of plasmid introduced and luminescence intensity in a two-hybrid assay equipped with the reporter gene (9096) of the present invention. Increasing the percentage of the plasmid expressing the reporter led to better ligand sensitivity. A method for measuring ligand activity based on the response speed of a reporter enzyme in a two-hybrid assay equipped with the reporter gene (9096) of the present invention. Comparison of luminescence rate when measured immediately after stimulation (0h) and when stimulated for 6 hours (6h). A method for measuring ligand activity based on the response speed of a reporter enzyme in a two-hybrid assay equipped with the reporter gene (9096) of the present invention. The rate of increase in the light emission rate when the stimulation time is 0, 3, 6, 18 hours is shown. Ligand selectivity in a two-hybrid assay using the reporter gene of the present invention. As a result of comparing the assay systems loaded with each mutant, the assay system expressing the mutant luminescent enzyme (8990 or 9096) of the present invention responds to female hormone (E2) rather than the conventional assay system expressing GLuc itself. Showed a stronger emission intensity. In the figure, “8990” is a GLuc mutant having a mutation in F89W / I90L, and “9096” is a GLuc mutant having a mutation in I90L / T96S. Ligand concentration dependency in a two-hybrid assay using the reporter gene of the present invention. Female hormone (E2) showed the strongest luminescence value for 10 ^-5 M stimulation. (A) Molecular structure of a conventional single molecule bioluminescent probe (SimGR3) based on GLuc and a single molecule bioluminescent probe (8990N) based on the high-intensity mutant. (Here, “8990N” represents a probe using the enzyme mutant “8990”. The same applies hereinafter.) (B) A conventional single molecule bioluminescent probe (SimGR3) based on GLuc Comparison of ligand sensitivity of single-molecule bioluminescent probe (8990N) based on luminance mutants. In the presence of the ligand, 8990N showed a better S / N ratio. (A) Examination of animal tissue permeability of bioluminescence by GLuc mutant (8990N) having mutation in F89W / I90L of the present invention. When the conventional GLuc itself and 8990N were expressed at the roots of the left back and right back of the mouse, it was found that bioluminescence from 8990N (right) was stronger than that of GLuc (left). (B) Observation of bioluminescence at the level of living animal tissue when mounted on a two-hybrid assay system. In addition to pACT-SH2 and pBIND-ER LBD, COS-7 cells were transplanted with transformed cells transfected with pG5-GLuc or pG5-90L, respectively, at the root of the left back of the mouse and right back of the mouse. Measured with or without. Stronger bioluminescence was observed from the tissue in which pG5-90L was introduced (right: GLuc expression of I90L mutant). (C) Comparison of relative luminescence values at each transplantation site of mouse in experiment (B). It corresponds to the sites (1), (2), (3), and (4) from the left. Comparison of bioluminescence properties using the Mann Marian two hybrid assay. It was confirmed that the ultra-bright bioluminescence enzyme (I90L or 8990) of the present invention was superior in luminescence intensity (absolute value) and S / N ratio than the conventional RLuc8.6-535. Comparison of bioluminescence characteristics using eukaryotic cells. It can be seen that the ultra-bright bioluminescence enzyme (I90L) of the present invention exhibits superior bioluminescence intensity compared to conventional M43I and RLuc8.6-535. Imaging of B16 skin cancer cells (melanoma) metastasis. Imaging comparison of cancer metastasis location when skin cancer cells expressing high-intensity bioluminescent enzyme (I90L) and conventional GLuc used in the present invention are respectively injected. Imaging of B16 skin cancer cells (melanoma) metastasis. Comparison of bioluminescence intensity for each organ by dissection. It can be seen that the luminescence values of the lungs and uterus are remarkably strong.

1. Modification of marine animal-derived luminescent enzyme gene used in the present invention (1) Marine animal-derived luminescent enzyme In the present invention, the term “marine animal luminescent enzyme” refers broadly to Gaussia, Renilla reniformis, Cypridina, In addition to Catria (Metridia) etc., the luminescent plankton companions Obelin, aqualine Pleuromanma, Oplophorus, etc. are also included, which refers to the luminescent enzyme (luciferase) produced by these “luminescent marine animals” , Gaussia luciferase (GLuc), and Caucasian luciferase (MLuc derived from Metridia longa, MpLuc1, MpLuc2, etc., a luminescent enzyme group derived from Metridia pacifica) have a very high distribution of hydrophilic and hydrophobic amino acids throughout each enzyme. These lucifers are similar and the amino acid sequence similarity of the putative enzyme active region is extremely high (FIG. 1). The hydrolase group also referred to simply as "marine animals light-emitting enzyme," but, also referred to as a particularly "Gaussia such luciferase". An amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more identity with the amino acid sequence of GLuc, MLuc, MpLuc1, or MpLuc2 and / or the base sequence encoding the amino acid sequence and The case encoded by the nucleotide sequence is also included. The Gaucian luciferases MLuc, MpLuc1, or MpLuc2 are slightly different in terms of molecular weight compared to GLuc, but in addition to the similarity in sequence as described above, the enzymatic properties such as substrate and luminescence activity are also GLuc. The following is a description of the typical Gaussia luciferase (GLuc). The knowledge gained in GLuc can be applied to other Gaussia luciferases. Hereinafter, unless otherwise specified, the indication of the modified position is represented by the position on the amino acid sequence of GLuc. For example, the 90th amino acid in GLuc corresponds to the 123rd amino acid in the MLuc amino acid sequence. In the case of MpLuc1 and MpLuc2, they correspond to the 114th and 93rd positions, respectively.
Gaussia luciferase (GLuc) has the following characteristics.
(A) Since it is the smallest bioluminescent enzyme discovered so far, when applied to molecular imaging, the burden on host cells and host proteins is much lighter.
(I) Extremely strong resistance to pH, surfactants and chemical modifiers.
(C) Highest brightness among bioluminescent enzymes.
(D) Turn-over of enzyme is fast. 7 times faster than conventional firefly luciferase.
On the other hand, bioluminescence generated by GLuc is easily affected by reaction conditions, and the drop in emission intensity is severe and unstable, so it is not suitable for use as a reporter (index) for emission analysis where stability is an important condition. It had been.

(2) Modification method for improving luminescent function (2-1) Improvement of luminescent function of marine animal luminescent enzyme and measuring method thereof In the present invention, when the function of marine animal luminescent enzyme is improved, it is important for observing reporter gene expression. The main aim is to increase the emission intensity and to improve the stability of the emission intensity over time, but also includes shifting the emission wavelength to a longer wavelength. Shifting to the longer wavelength side increases permeability from cells, skin, etc., which is an important property for expanding the use of reporter genes.
The emission intensity can be obtained by measuring the emission intensity in a specific wavelength region using a conventional emission spectrophotometer after the addition of the substrate, so that two-dimensional information can be obtained. It can be measured. A light-emitting plate reader can also be used, and more accurate data can be acquired because of excellent sample processing ability. At that time, the area of each emission spectrum is measured after sufficient lysis time (about 20 minutes). Further, the shift to the long wavelength side can be measured by a wavelength scanning method or by providing a long wavelength filter.

(2-2) Modification Method In order to modify the amino acid sequence of the marine animal luminescent enzyme of the present invention, a desired amino acid residue of the amino acid sequence itself can be chemically changed. In this case, the base corresponding to the amino acid to be substituted in the base sequence encoding the enzyme may be point mutated. As a base mutation method, a known method such as a site mutation method can be appropriately used. In the embodiment of the present invention, as described in the previous patent application (Japanese Patent Application No. 2009-101025), the point mutation was introduced according to the “quick change method” (Non-patent Document 10), but this is not limitative.

(2-3) Production of Mutant Luminescent Enzyme and Analysis of Luminescent Function The present inventors have filed an earlier patent application (patent application) against GLuc, which is one of marine animal luminescent enzymes. Based on the modification strategy described in 2009-101025), one or more of the amino acid residues constituting the enzyme active region (GLuc positions 89 to 118) are “conservative amino acid replacement”. ) ”Was made.
Specifically, for GLuc, which is a typical example of Gaussia luciferase, we investigated a strategy for introducing a mutation that results in substitution of conservative amino acids at 18 positions in positions 89 to 118 that are presumed to be active sites. Then, DNAs encoding 57 types of GLuc mutants were prepared (FIG. 2A). The pcDNA3.1 (+) vector inserted with the GLuc mutant DNA was introduced into COS-7 cells cultured on a 12-well plate, Incubated for 16 hours. Subsequently, COS-7 cells in each well were immersed in a cell lysis buffer for 5 minutes to prepare a cell lysate, and the spectrum in the presence of a substrate specific to the luminescent enzyme (coelenterazine) was measured using a fluorometer (F- 7000, Hitachi) and the effectiveness of the introduced mutation was examined (FIG. 2B-1). FIG. 2B-2 shows the case where the time of immersion in the cell lysate is 20 minutes and the area of each emission spectrum is measured with a luminescence plate reader. From the fact that GLuc itself before modification was luciferase showing particularly strong luminescence intensity among luciferases, the intensity of luminescence intensity of these mutant luminescence enzymes used in the present invention can be seen.
In addition, the emission spectra of Y97W, I90L, F89W / I90L / H95E / Y97W (Mon3) compared with the conventional GLuc are shown in FIG. 2D, and bioluminescence in the long wavelength region using a 610 nm long-pass filter is shown. From the relative intensity of intensity (FIG. 2E), it can be seen that various mutants exhibit strong bioluminescence on the long wavelength side. “Mon3” not only increased the emission intensity, but a long wavelength shift of 35 nm was observed from the absorption wavelength of the original GLuc. Such long-wavelength bioluminescence has high utility value for use as an excellent analysis signal in the future because the tissue permeability of the living body is improved.
Furthermore, in order to check the stability of the bioluminescence intensity, a substrate (dissolved in PBS buffer) was introduced into each mutant, and the change in bioluminescence with time was observed. When observed for 5 seconds for I90L, H95E, and Y97W, they show relatively stable luminescence intensity (Fig. 4A). The result of observation for a longer time (10 minutes) shows that the decrease in the original GLuc luminescence value (white circle) is severe. On the other hand, the emission values of I90L (black square) and I90V (gray triangle) showed relatively stable emission intensity (FIG. 4B). The results of observing the change in bioluminescence over time for a short time (5 seconds) after introducing a substrate (dissolved in Matthew modified buffer) into each mutant were the same (FIG. 4C). Moreover, after introducing a substrate (dissolved in Matthew modified buffer) into each mutant stepwise, the results of observing the change in bioluminescence over time for each step for one second (FIG. 4D) are also very high. It can be seen that it shows stable and strong bioluminescence.
As a substrate for the GLuc mutant, coelenterazine (CTZ), which is the standard substrate for GLuc, is preferable in that it exhibits high emission intensity, but by changing the type of coelenterazine, the wavelength at which the peak value is shown is slightly shifted. (Prior Patent Application No. 2009-101025).

(2-4) Variants of other Gaussia luciferases Gausia luciferases derived from caussia (Metridia pacifica or Metridia longa), which are Gausia marine animals very similar to Gaussia, specifically MpLuc1 (from Metridia pacifica) and In MLuc (from Metridia longa) luciferase, a mutant (MpLuc1-I114L mutant) due to conservative amino acid substitution at position 114 of MpLuc1, corresponding to position 90 of the amino acid sequence of GLuc, and conservative at position 123 of MLuc MpLuc1 Y113W / I114L / H119E / Y121W mutant (MpLuc4) and MLuc Y122W corresponding to the GLuc F89W / I90L / H95E / Y97W (Mon3) mutant as well as mutants due to amino acid substitution (MLuc-I123L mutant) / I123L / H128E / Y130W mutant (MLuc4) was prepared, and its luminescence characteristics were measured. As in the case of the GLuc variant, bioluminescence intensity increased, stability increased, and the wavelength shifted to longer wavelengths. Remarkable effect is achieved (FIGS. 5A ~ C, FIG. 6A ~ D), findings in GLuc was confirmed to be applicable also to all other Gaussia class luciferase.

(2-5) Mutant luminescent enzyme used in the present invention with improved luminescent function From the above results, in marine animal luminescent enzyme (Gaussia luciferase), amino acid sequence 89th, 90th, 95th, 97th in GLuc, Luminescence of the luminescent enzyme is increased by applying “conservative amino acid replacement” to one or more amino acid residues corresponding to positions 100, 108, 112, 115, or 118. At the same time, it is possible to achieve an improvement in the function of the luminescent enzyme activity in which the luminescence intensity is stabilized or a shift to a longer wavelength occurs. Specifically, when the hydrophobic amino acid residues corresponding to the 89th, 90th, 97th, 108th, 112th, 115th, or 118th position are mutated to other hydrophobic amino acid residues, respectively. In addition, the function of luminescent enzyme activity is improved such that the luminescence intensity increases or shifts to a longer wavelength. In particular, mutations from hydrophobic and nonpolar amino acid residues at positions 90, 108, 112, 115, or 118 to other hydrophobic and nonpolar amino acid residues, or position 89 or By causing a mutation from the aromatic amino acid residue at position 97 to another aromatic amino acid residue, the intensity of the luminescent enzyme is increased and stabilized, and a shift to a longer wavelength occurs. In addition, a shift to a longer wavelength occurs due to a mutation from a hydrophilic amino acid residue that forms a hydrogen bond at the 95th or 100th position to another hydrophilic amino acid residue.
That is, when the mutant luminescent enzyme having an improved luminescent function in the present invention is used, in the marine animal luminescent enzyme (Gaussia luciferase), at least one of amino acid residues at positions corresponding to amino acid positions 89 to 118 in GLuc. Wherein the substitution is at least at a position corresponding to position 89, 90, 95, 97, 100, 108, 112, 115, or 118. Substitutions that include conservative amino acid replacements for amino acid residues at positions selected from one are included.
Preferably, a hydrophobic and nonpolar amino acid residue at position 90, 108, 112, 115, or 118 includes a mutation to another hydrophobic and nonpolar amino acid residue, or the same Others from the hydrophilic amino acid residue containing a mutation from the aromatic amino acid residue at

position

89 or 97 to another aromatic amino acid residue or forming a hydrogen bond at position 95 or 100 Including a mutation to a hydrophilic amino acid residue.
More preferably, the position corresponding to position 89 on the amino acid sequence of GLuc is tryptophan, position 90 is leucine or valine, position 95 is glutamic acid, position 97 is tryptophan, position 108 is valine, position 112 is leucine. Alternatively, substitution of valine, position 115 with valine, or position 118 with leucine or valine shows particularly significant emission intensity enhancement and also a long wavelength shift. In particular, leucine mutants at position 90 (GLuc-I90L, MpLuc1-I114L, MpLuc1-I114L) are high-intensity luminescent enzymes that have not only high brightness but also remarkable long-wavelength shift. In addition, mutants to glutamine or asparagine, which are hydrophilic amino acids that form hydrogen bonds at positions 95 and 100, cause a long wavelength shift.
Not only such a single amino acid mutant but also two or more positions can be subjected to the same substitution, so that a higher light emission function can be provided. Specifically, the isoleucine at the position corresponding to position 90 on the amino acid sequence of GLuc was substituted with leucine, and at the same time, tryptophan substitution at position 89 or leucine substitution at position 115 (GLuc- F89W / I90L, or GLuc-I90L / I115L) and four mutants (Glucuc) in which substitution of leucine at position 90, substitution of tryptophan at position 89, substitution of glutamic acid at position 95 and tryptophan substitution at position 97 were performed simultaneously -F89W / I90L / H95E / Y97W (Mon3), MpLuc-Y113W / I114L / H119E / Y121W (MpLuc4) and MLuc-Y122W / I123L / H128E / Y130W (MLuc4)) all show significant emission intensity and long It is an epoch-making high-intensity luminescent enzyme that also causes a remarkable shift to the wavelength side.
In the present invention, when “mutant luminescent enzyme (modified luciferase)” is used, the amino acid sequence corresponding to positions 89 to 118 on the amino acid sequence of GLuc, which is a region corresponding to the active center, is in principle It is desirable that the amino acid sequence of the original Gaussia luciferase has no mutation other than the above-mentioned “similar amino acid substitution”. However, as long as it is a sequence other than the region, a mutation within the range in which the three-dimensional structure of the whole enzyme does not change greatly is acceptable, and in particular, a part of the N- or C-terminal portion, for example, 1 to 50, preferably 1 to 30, More preferably 1 to 20, even more preferably 1 to 10 amino acid residues are deleted. Specifically, in the amino acid sequence of the modified Gaussia luciferase, the amino acid sequence excluding the region corresponding to positions 89 to 118 on the GLuc amino acid sequence is 70% or more, preferably 80% or more, more preferably Can be expressed as an amino acid sequence having 90% or more, most preferably 95% identity.

(2-6) Change of mutant luminescent enzyme gene to mammal compatible codon As described later, when the mutant luminescent enzyme gene of the present invention is used as a reporter gene, typically, a reporter gene assay method or a two-hybrid method is used. A series of operations for producing a vector by combining the mutant luminescent enzyme gene of the present invention with a promoter of a target gene, introducing it into a host cell, and culturing it. All known procedures can be applied.
The mutant luminescent enzyme gene used at that time is preferably modified to a base sequence that is easily expressed in the host cell by changing it to a codon suitable for the cell depending on the host cell used. In addition, it is necessary to appropriately perform modification to provide a restriction enzyme site for insertion into a vector. A well-known method can also be applied to these steps, but as an example, when used in a living body of a mammal such as a human, or a modified body assumed to be used in a mammalian cell in vitro, FIG. 7 (A), ( Shown as B). The function improvement modification strategy at that time is described in detail in the previous application (Japanese Patent Application No. 2009-101025).

3. Analytical systems using reporter genes Reporter genes have been used for analysis of gene expression timing and expression levels when recombinant proteins are produced by recombinant DNA technology, especially in response to external stimuli. It is widely used as an index indicating the time and expression level change. In the present invention, an analysis system using a reporter gene is typically a reporter gene assay, a two-hybrid assay, etc., but also includes PSA (protein splicing assay), PCA (protein complementation assay), fluorescence anisotropy, etc. It is. Hereinafter, a reporter gene assay and a two-hybrid assay, which are typical analysis systems, will be described in detail.

(1) Reporter gene assay The reporter gene assay method is frequently used as an analysis means of transcription factor activation and gene expression regulation by external stimulation, but typically disrupts signal transduction via nuclear receptors. Used to detect endocrine disruptors (environmental hormones). The expression of a target gene (eg, a hormone responsive gene) related to signal transduction through a nuclear receptor is expressed by a cis region (hormone response element) in which a complex of a ligand and a receptor regulates transcription of the gene. ). This is a method for detecting the amount of hormone molecules or endocrine disrupting substances that can serve as ligands based on the amount of bioluminescence, etc., by introducing a plasmid incorporating a reporter gene such as luciferase downstream of the cis region of these various hormone-responsive genes. .
As the host cell in this case, COS cells, CHO-K1 cells, HeLa cells, HEK293 cells, NIH3T3 cells, etc. of mammalian cells used for general gene recombination are preferably used. Bacteria such as yeast cells and Escherichia coli Although it may be a cell, an insect cell, etc., it is often used in a living body of a mammal including human beings or in a mammalian cell in vitro. In that case, it is preferable that the mutant luminescent enzyme gene of the present invention is modified to a base sequence that can be easily expressed in the host cell by changing to a codon suitable for the cell depending on the host cell to be used. In addition, it is necessary to appropriately perform modification to provide a restriction enzyme site for insertion into a vector, and well-known methods can be applied to these steps.

In the case of firefly luciferase, which has been widely used in reporter gene assay methods, (a) the molecular weight is large and it takes time until expression, which places a heavy burden on the host cell. Since it is low, all the disadvantages that it is usually necessary to wait for 1-2 days after stimulation before a sufficient amount of luciferase (reporter) accumulates is eliminated.
By using the mutant luminescent enzyme of the present invention, the luminescence intensity of the reporter is extremely high (especially in the case of the I90L mutant, 14,000 times stronger than the conventional firefly luciferase), so it can be measured in a very short time after stimulation. Therefore, the measurement time can be significantly shortened compared to the prior art, and the stability of luminescence over time is high, so that luminescence can be measured even in cell lines with poor gene transfer efficiency. Further, since the shift to the long wavelength side increases the permeability through the cell membrane and the skin, the background value decreases and the measurement accuracy is high.
Specifically, in order to apply the mutant luminescent enzyme of the present invention to these reporter gene assays, the luminescent enzyme is linked to a known eukaryotic expression vector carrying an expression promoter suitable for the assay upstream. After introduction into a eukaryotic cell and after a certain period of time, it may be used for measurement under the condition that there is no signal (stimulation) (Non-patent Document 20). As an expression vector for reporter gene assay that can be equipped with the luminescent enzyme, a known pTransLucent vector can be used and can be easily mounted using a known method.

(2) Two-hybrid method The two-hybrid method (Two-hybrid method) is one of the methods to investigate the interaction between proteins. In 1989, the yeast two-hybrid (Y2H) system using yeast (Saccharomyces cerevisiae) was used. Was first built. GAL4DBD and any protein A (bait) can be expressed as a fusion protein using the fact that the DNA-binding domain (DBD) and transcriptional activation domain of the transcriptional activator GAL4 protein are separable. It can be determined whether or not it interacts with the transcription activation domain (TA) expressed in (1) and protein B (prey) as a fusion protein. When the proteins A and B bind to each other, DBD and TA are close to each other and the DNA binding domain (DBD) binds to the “UASG” base sequence, so that expression of a reporter gene linked downstream thereof is promoted. If the reporter gene is luciferase, the affinity of both A and B proteins can be measured by monitoring bioluminescence in the presence of the specific substrate, and screening for proteins and peptides that interact with protein A (bait) Can do. Protein B (prey) at that time can also be provided by an expression library.
Host cells are not limited to yeast cells, and bacteria such as E. coli, mammalian cells, and insect cells are also used. In this case, in addition to the DNA binding domain (DBD) of GAL4, which is a transcriptional activator derived from yeast, “LexA” of a repressor protein derived from E. coli can also be used. A DNA encoding these and a DNA encoding a bait protein (that is, the above-mentioned arbitrary protein A) such as a ligand-binding region of a ligand-responsive transcriptional regulator are ligated downstream of a promoter that can function in the host cell. Link. On the other hand, as the “transcription activation region of transcription activator”, for example, the transcription activation region of GAL4, the B42 acidic transcription activation region derived from E. coli, the transcription activation region of herpes simplex virus VP16, etc. can be used. . A DNA encoding these transcriptional activation regions and a DNA encoding a prey protein (that is, the above-mentioned arbitrary protein B) are linked and downstream of a promoter that can function in the host cell. Specifically, for example, the plasmid pGBT9 (manufactured by Clontech) can be used as a vector having DNA encoding the DNA binding region of the transcriptional regulatory factor GAL4 and allowing budding yeast to be used in host cells. As a vector having DNA encoding a transcription activation region and usable in budding yeast, plasmid pGAD424 (manufactured by Clontech) and the like can be mentioned. Examples of vectors that have a DNA encoding the DNA binding region of GAL4 and can be used in mammalian cells include pM (Clontech), pBIND (Promega), etc., and herpes simplex virus VP16 Examples of vectors having a DNA encoding a transcription activation region and usable in mammalian cells include pVP16 (manufactured by Clontech), pACT (manufactured by Promega), and the like. In addition, examples of vectors that have DNA encoding the DNA binding region of LexA and can be used in mammalian cells include pLexA (manufactured by Clontech) and the like. Examples of vectors that can be used in the above include pB42AD (manufactured by Clontech). As a kit for a two-hybrid system, Matchmaker Two-hybrid System (manufactured by Clontech), CheckMate Mammalian Two-Hybrid System (Promega), etc. are already on the market and are incorporated into the kit so that firefly luciferase is expressed. ing.
In addition, a gene encoding the mutant luminescent enzyme of the present invention may be constructed by inserting a vector inserted as a reporter gene downstream of a region to which GAL4 binds (“UASG”). For example, in the case of a mammalian host, a commercially available pG5Luc vector (Promega) or pFR-Luc vector (Stratagene) can be used instead of firefly luciferase mounted on the vector. It can also be used in place of the commercially available pG5CAT vector (Clontech) Chloramphenicol acetyltransferase (CAT).

4). New measurement method in an analysis method using a reporter gene Since the brightness of the mutant luminescent enzyme of the present invention is extremely high and stable, a new kinetics applicable to various analysis methods using a reporter gene in the present invention is achieved. Based on the measurement method developed.
The reporter gene assay of the present invention is the same as the conventional reporter gene assay method until the reporter protein is expressed in response to an external stimulus. However, as a method for measuring the enzyme activity of the expressed reporter gene, the present inventor Devised a measurement method based on kinetics as follows.
(1) A plasmid containing the mutant luminescent enzyme of the present invention as a reporter gene is introduced into a cell and cultured in a well.
(2) A test substance (hormone or the like) is added, and after a certain period of time, a lysis solution (lysis) is added to sufficiently lyse cells (create a lysate).
(3) The well plate is set in a plate reader, and after adding the substrate, the intensity of response stimulus of the test substance is observed from the rate of increase in luminescence per unit time (ie, RLU / sec).
(4) The rising speed is measured from the difference between the initial light emission value and the light emission value after a certain period of time.
The rising speed v can be expressed as follows.
v = (RLU ₁ -RLU ₀ ) / (t ₁ -t ₀ )
Here, RLU ₀ indicates the initial light emission intensity (relative luminescence unit; RLU), and RLU ₁ indicates the light emission intensity after elapse of a predetermined time (t ₁ ).
Thus, the measurement method of the present invention uses the light emission response speed as an analysis signal.

5. Others Other terms and concepts in the present invention are defined in detail in the description of the embodiments and examples of the present invention. The terms are basically based on the IUPAC-IUB Commission on Biochemical Nomenclature, or based on the meanings of terms commonly used in the field. Various techniques used for carrying out the invention can be easily and surely implemented by those skilled in the art based on known literatures and the like, except for the techniques that clearly indicate the source. For example, genetic engineering and molecular biology techniques are described in J. Sambrook, EFFritsch & T. Maniatis, “Molecular Cloning: A Laboratory Manual (2nd edition)”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); DMGlover et al.ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); Ausubel, FM et al., Current Protocols in Molecular Biology , John Wiley & Sons, New York, NY, 1995; Japan Biochemical Society, “Sequel Biochemistry Experiment Course 1, Genetic Research Method II”, Tokyo Chemical Doujin (1986); Japan Biochemistry Society, “New Biochemistry Experiment Course 2” , Nucleic Acid III (Recombinant DNA Technology) ”, Tokyo Kagaku Dojin (1992); R. Weed.,“ Methods in Enzymology ”, Vol. 68 (Recombinant DNA), Academic Press, New York (1980); R. Wu et al.ed., "Methods in Enzymology", Vol. 100 (Recombinant DNA, Part B) & 101 (Recombinant DNA, Part C), Academic Press, New York (1983); R. Wu et al.ed., "Methods in Enzymology", Vol.153 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E) & 155 (Recombinant DNA, Part F), Academic Press, New York (1987), etc., the methods described in the literature cited therein, or methods substantially similar to them or modification methods. In addition, various proteins and peptides used in the present invention or DNAs encoding them can be obtained from existing databases (URL: http://www.ncbi.nlm.nih.gov/ etc.).

EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further in detail and concretely, this invention is not limited to the following examples.
In addition, the description content in the technical literature, the patent gazette, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.

[Example 1] Reporter gene assay loaded with GLuc mutant In order to prove the usefulness of the analysis method using the GLuc mutant developed by the present inventors, pG5Luc (an example of a conventional reporter gene assay system) A new reporter gene assay system in which Promega) was loaded with the GLuc mutant was constructed (FIG. 8). First, a vector having an androgen response element (ARE) upstream was prepared, and a gene encoding the conventional GLuc itself or the GLuc mutant of the present invention was linked downstream thereof by genetic engineering (FIG. 8A). This new vector was introduced into COS-7 cells and stimulated for 16 hours in the presence or absence of androgen. Thereafter, cell lysates were prepared, a specific substrate (coelenterazine) was added, and the difference in bioluminescence value was immediately measured with a plate reader.
As a result, compared to the conventional reporter gene assay system equipped with GLuc, the luminescence intensity change was stronger when the GLuc mutant (190L) of the present invention was loaded (FIG. 8B). As an analysis method of the luminescence response, by taking an area from substrate introduction to a certain time (1 second), the biological activity of the male hormone could be observed with high luminance (FIG. 8C).

[Example 2] Two-hybrid assay loaded with GLuc mutant In order to prove the usefulness of the analytical method using the GLuc mutant, a Manmarian two-hybrid assay system loaded with the GLuc mutant was constructed, An experiment to measure the interaction between proteins was performed (FIG. 9).
In order to carry out this experiment, the following plasmid groups were first prepared based on the conventional typical two-hybrid assay plasmids pACT, pBIND and pG5Luc: (1) plasmids encoding ER LBD and Gal4; (2) Plasmid encoding SH2 domain and VP16, (3) pG5 plasmid carrying conventional GLuc or high-intensity mutant (I90L) of the present invention as a reporter gene.
When the plasmids (1), (2) and (3) were co-introduced into COS-7 cells, stimulation was performed for 6 hours under the condition of female hormones, and changes in luminescence values were observed. The two-hybrid assay loaded with the mutant (I90L) showed a stronger luminescence value and S / N ratio (FIG. 9B). Moreover, the kinetics showed the same result (FIG. 9C, D).
Furthermore, in order to observe the luminescence value dependent on the female hormone stimulation time, the following plasmid groups were first prepared based on the conventional typical two-hybrid assay plasmids pACT, pBIND and pG5Luc: (1) ER A plasmid encoding LBD and Gal4, (2) a plasmid encoding SH2 domain and VP16, and (3) a pG5 plasmid carrying a conventional GLuc or the high-intensity mutant of the invention (9096) as a reporter gene.
After cells (1), (2) and (3) were co-introduced with the solvent itself (0.5% DMSO) or female hormone (estrogen) stimulation for 0, 3, 6, 18 hours, The bioluminescence value was measured with a luminescence photometer (FIG. 9E). As a result, the cells carrying the plasmid carrying the GLuc mutant of the present invention showed an excellent S / N ratio against female hormone stimulation. For example, when the GLuc mutant was loaded, the luminescence value increased up to 2 times after 3 hours of stimulation, but conventional GLuc could not be distinguished. This result shows that the GLuc mutant of the present inventors has a high brightness, and thus a strong luminescence increase that can be distinguished even when stimulated for a significantly shorter time than that of the conventional case (ie, when a small amount is expressed) is observed. did it.

[Example 3] Optimization of plasmid introduction ratio in two-hybrid assay
Usually, in order to carry out a Mammalian two-hybrid assay, three plasmids (a plasmid expressing proteins X and Y and a plasmid having a response sequence that recognizes the bond between XY) are introduced into the same cell. At this time, the ratio of the plasmid introduced into the cell is an important factor that determines the efficiency of the assay. In this example, the optimum ratio was examined in performing the assay by changing the introduction ratio of each plasmid. First, pBIND that expresses ER LBD and pACT plasmid that expresses Src SH2, and a pmG5 plasmid that expresses a reporter protein in response to the binding between ER LBD and SH2 were prepared. Next, the introduction ratios of the three types of plasmids were changed and introduced into COS-7 cells, and the difference in luminescence value was measured under the condition with and without female hormone stimulation.
The result is shown in FIG. According to the results, when the ratio of pACT: pBIND: pmG5 was 1: 1: 3, the best signal-to-background ratio (S / N ratio) was shown. As an overall trend, it was observed that the higher the ratio of pmG5, the better the S / N ratio.

[Example 4] Measurement of hormone sensitivity based on kinetics
In the reporter gene assay of the present invention, a reporter protein is expressed in response to an external stimulus. As a method for measuring the enzyme activity of the expressed reporter, the present inventors have devised the following measurement method.
(1) First, eukaryotic cells are cultured in a 96-well plate, and then the plasmid of the present invention is introduced into the cells. At this point, the assay preparation is complete.
(2) Next, a female hormone or chemical substance is added to each well of a 96-well plate, and after a certain period of time, a lysate solution (lysis) is added to prepare a lysate.
(3) A 96-well plate is set in a plate reader, and after the addition of a substrate, the intensity of hormone stimulation is observed from the rate of increase in luminescence per unit time (ie, RLU / sec).
As shown in FIG. 11 (A), when COS-7 cells are used, the rising tendency of the initial luminescence value is greatly different depending on the presence or absence of female hormone stimulation. The concentration and nature of female hormones can be observed from this initial velocity. At this time, the rising speed v can be expressed as follows.
v = (RLU ₁ -RLU ₀ ) / (t ₁ -t ₀ )
Here, RLU ₁ and RLU ₀ indicate the emission intensity at a certain time point and the initial emission intensity (relative luminescence unit; RLU), respectively.
According to this calculation method, when stimulated for 0 h (control), it was 28.5 RLU / sec, and when stimulated for 6 h, it was 76.5 RLU / sec. From this result, it can be seen that the light emission response time can be used as an analysis signal.

Fig. 11 (B) shows the result of a more detailed investigation. That is, a two-hybrid system equipped with 9096, which is one of the high-intensity mutants according to the present invention, was created. For this purpose, pG5-9096 expressing 9096 was co-introduced together with pBIND-ER LBD and pACT-SH2 into COS-7 cells cultured in 96-well plates. Thereafter, expression of the reporter protein was induced according to the stimulation time of the female hormone. Thereafter, a cell lysate was prepared, and the increase in luminescence value before and after substrate introduction was measured. The rate of increase in bioluminescence per unit time (RLU / sec) is 33.3 RLU / sec (0 hour stimulation), 43.3 RLU / sec (3 hour stimulation), 96.7 RLU / sec (6 hour stimulation), 150.0 RLU / sec (18 hour stimulation). It was found that the rate of increase in emission intensity dramatically increases with each stimulation time.

[Example 5] Ligand selectivity of the two-hybrid assay In order to verify the ligand selectivity of the two-hybrid assay, various pG5 plasmids carrying high-intensity mutants were prepared and female hormone sensitivity was measured. (FIG. 12). For this experiment, COS-7 cells were cultured on a plate, and in addition to pBIND-ER LBD and pACT-SH2, any of pG5-GLuc, pG5-I90L, pG5-8990, and pG5-9096 was added to COS-7. Co-introduced into cells. Thereafter, the difference in luminescence value was measured under the condition of female hormone (E2) or its antagonist (OHT).
As a result, as shown in FIG. 12, it was found that stronger bioluminescence can be ensured when the GLuc mutant of the present invention is mounted than when the conventional GLuc itself is mounted. It was also found that E2 promotes ER LBD-SH2 binding and induces strong bioluminescence compared to OHT.

[Example 6] Ligand concentration dependency of the two-hybrid assay The following experiment was conducted to examine the ligand concentration dependency of the two-hybrid assay (Fig. 13). COS-7 cells were cultured on a cell culture plate, and pBIND-ER LBD, pACT-SH2, and pG5-8990 were simultaneously introduced. After 12 hours of stimulation with various concentrations of ligand, changes in the luminescence values were observed.
As a result, it was found that the binding between ER LBD-SH2 strongly responded to female hormone stimulation (ie, had ligand selectivity), and the concentration reached 10 ⁻⁵ M, and the luminescence intensity reached the maximum value.

[Example 7] Bioluminescent probe using the mutant luminescent enzyme of the present invention In order to prove the superiority of the high-luminance bioluminescent enzyme of the present inventors, a novel bioluminescent probe using this enzyme was prepared ( 8990N). As a control, a probe based on the conventional GLuc itself was also synthesized (SimGR3). The molecular structure of each probe is shown in FIG. That is, each probe contains a stress hormone receptor ligand-binding domain (glucocorticoid receptor ligand binding domain). This makes them stress hormone sensitive.
As a result of investigating the stress hormone responsiveness of each probe, it was found that 8990N showed stronger bioluminescence, and also produced a bioluminescence value that could be sufficiently separated under the condition with or without ligand stimulation (FIG. 14B, □). And ■). ■ indicates the case of stress hormone stimulation. On the other hand, SimGR3 could not distinguish stress hormone stimulation significantly (○ and ●).

[Example 8] Biological imaging using GLuc mutant The improvement (high brightness, long wavelength shift, stability, etc.) of the GLuc mutant of the present invention was utilized to verify its applicability to biological systems ( FIG. 15).
First, a pcDNA3.1 (8990) plasmid inserted with a gene encoding 8990, a high-intensity mutant of GLuc, was constructed and introduced into COS-7 cells. On the other hand, as a control, pcDNA3.1 (GLuc) plasmid into which a gene encoding conventional GLuc itself was inserted was similarly introduced into COS-7 cells. Thereafter, both transformed cells were transplanted into the left and right upper / subcutaneous tissues of BALB / c nude mice (five weeks old female). After waiting for 12 hours until the transplanted cells were stabilized, the same amount of substrate (coelenterazine) was injected, and then the degree of the luminescence value was compared (FIG. 15A). As a result, stronger bioluminescence could be observed from the site where the transformed cells expressing 8990 were transplanted.

Furthermore, a living cell equipped with a mammalian two-hybrid system expressing the GLuc mutant was prepared, and a biological imaging system using the same was tried (FIG. 15B).
First, plasmids were constructed by connecting the female hormone receptor (ER LBD) and Src SH2 domains to BIND and ACT, respectively (pBIND and pACT). On the other hand, as a response system, a plasmid having a Gal4 response element upstream and expressing I90L was also prepared (pG5-I90L). As a control, a plasmid expressing the conventional GLuc itself was prepared (pG5-GLuc).

The three types of plasmids (pBIND, pACT, pG5-I90L or pG5-GLuc) were co-transfected into COS-7 cells to produce transformed cells. The transformed cells were transplanted into the left and right upper and subcutaneous tissues of BALB / c nude mice (five-week-old female), respectively. At this time, the left upper part was transplanted with control (pG5-GLuc), and the right upper part was transplanted with transformed cells having pG5-I90L. Furthermore, 12 hours were allowed for the transplantation to stabilize.
Later, female hormone or solvent (0.1% DMSO) was injected into each mouse, and the luminescence intensity was observed after another 6 hours (FIG. 15B). As a result, mice subjected to female hormone stimulation showed stronger bioluminescence. Also, in the mouse stimulated with female hormones, the right upper (I90L expression) showed stronger bioluminescence than the left upper (GLuc expression). Even if the same amount of GLuc or I90L is expressed by female hormone stimulation, it can be analyzed that I90L showed higher bioluminescence because it was more stable and brighter. The specific light emission intensity is shown in FIG.

[Example 9] Comparison of bioluminescence characteristics using the Manmarian two-hybrid assay Further, when the bioluminescent enzyme (I90L, 8990) is mounted on the mammalian two-hybrid assay, the known RLuc The case of mounting the mutant RLuc8.6-535 was compared (FIG. 16). RLuc8.6-535 (Non-patent Document 11) used here is the brightest artificial bioluminescent enzyme developed so far, and is A55T / C124A / S130A / K136R / A143M / M185V / M253L / S287L. It is an RLuc mutant having mutations at multiple locations, / A123S / D154M / E155G / D162E / I163L / V185L. As an experimental method in this experiment, a system that visualizes the binding between the ER LBD and the Src SH2 domain was used in the same manner as the method shown in Example 2. As a result, it was confirmed that the bioluminescence value (absolute value) of 8990 was 11.4 times stronger than that of RLuc8.6-535 as a result of the experiment under the same conditions. As for the S / N ratio, 8990 showed an S / N ratio that was about twice as good as that of RLuc8.6-535.
This result shows that the two-hybrid assay equipped with the GLuc mutant enzyme of the present invention is superior to the assay system loaded with other luminescent enzymes.

[Example 10] Comparison of bioluminescence characteristics using eukaryotic cells The difference in luminescence value between the GLuc mutant enzyme according to the present invention and the conventional bioluminescence enzyme was measured (Fig. 17). First, plasmids expressing the respective bioluminescent enzymes were introduced into eukaryotic cultured cells (COS-7) and then cultured for 16 hours. Thereafter, the cells were collected and a lyset was prepared. The luminescence value after substrate introduction was measured immediately with a luminometer. The luminescence values of I90L of the GLuc mutant enzyme developed by the present inventor, the M43I mutant (Non-patent Document 12), which is a known GLuc mutant, and RLuc8.6-535 were compared. As a result, I90L according to the present invention exhibited a bioluminescence that was 5 to 20 times stronger than conventional M43I and RLuc8.6-535.

[Example 11] Imaging of in vivo metastasis of B16 skin cancer cells (melanoma) Taking advantage of the GLuc mutant enzyme exhibiting high-intensity bioluminescence used in the present invention, cancer cell metastasis experiments in living mice The results of (metastesis) are shown in FIG. For this experiment, B16 skin cancer cells (melanoma) were first introduced with the conventional GLuc gene and the I90L gene of the high-intensity bioluminescent enzyme of the present invention and cultured for 16 hours. Thereafter, the cells were collected and intravenously injected into living 6-week-old BALB / c nude mice. Thereafter, the substrate was similarly injected into the vein of the mouse, and the bioluminescence was observed with a small animal imaging device (IVIS Lumina XR (Xenogen)).
As a result, the bioluminescence intensity in the mouse body was very intense in the lung region (FIG. 18A). At this time, especially the mice injected with cells expressing I90L selectively show strong bioluminescence, so the GLuc mutant enzyme used in the present invention observed the cancer metastasis process in vivo. It turned out to be superior in doing.
Further, from the result of dissection and imaging of the internal organs, it was confirmed that strong bioluminescence was shown in the lung and uterus (FIG. 18B). This result confirms that the bioluminescence observed in FIG. 18A is due to cancer cells that have metastasized to the lungs or uterus. This result also shows that the GLuc mutant enzyme of the present invention can exhibit very excellent performance in biological imaging, particularly in observation of the presence or absence of cancer tissue, cancer cell metastasis, and the like.

Claims

A reporter gene used in an analysis method for analyzing the timing or amount of expression of a target gene in a cell in response to an external stimulus, the amino acid sequence of Gaussia luciferase (GLuc) in the amino acid sequence of marine animal luminescent enzyme At least one of the amino acid residues at positions corresponding to positions 89 to 118 above is substituted, and the substitution is the same at positions 89, 90, 95, 97, 100, 108, Mutation luminescence with conservative amino acid replacement for amino acid residues at positions selected from at least one of positions corresponding to positions 112, 115, and 118, thereby improving luminescence function A reporter gene comprising an enzyme-encoding gene.
The gene encoding the mutant luminescent enzyme is a position corresponding to position 89, 90, 97, 108, 112, 115, or 118 on the amino acid sequence of GLuc in the amino acid sequence of marine animal luminescent enzyme From one of the hydrophobic amino acid residues to another hydrophobic amino acid residue, or from one hydrophilic amino acid residue that forms a hydrogen bond at the 95th or 100th position to another hydrophilic amino acid residue The reporter gene according to claim 1, which encodes a mutant luminescent enzyme having a conservative amino acid substitution comprising:
In the amino acid sequence of the marine animal luminescent enzyme, the amino acid residue at the position corresponding to the 90th, 108th, 112th, 115th, or 118th position on the GLuc amino acid sequence is replaced with leucine (L ), Substituted with tryptophan (W) or valine (V), or substituted with tryptophan (W) for the amino acid residue at the position corresponding to position 89 or 97, or at the position corresponding to position 95 The reporter gene according to claim 2, comprising substituting an amino acid residue with glutamic acid (E) or substituting an amino acid residue at a position corresponding to position 100 with asparagine (N).
In the amino acid sequence of the marine animal luminescent enzyme, the gene encoding the mutant luminescent enzyme is subjected to a conservative amino acid substitution including substituting an amino acid residue at a position corresponding to position 90 on the amino acid sequence of GLuc with leucine. The reporter gene according to any one of claims 1 to 3, which is a mutant luminescent enzyme gene.
The reporter gene according to any one of claims 1 to 4, wherein the marine animal luminescent enzyme is any luminescent enzyme selected from Gaussia luciferase (GLuc) and Caucasian luciferase (MLuc, MpLuc1, and MpLuc2). .
An expression vector comprising the reporter gene according to any one of claims 1 to 5 downstream of a response sequence to an external stimulus or a response sequence to a fusion protein formed by a response to the external stimulus.
A transformed cell into which the expression vector according to claim 6 has been introduced.
An analysis method for analyzing the timing or expression level of a target gene in a cell in response to an external stimulus, wherein the transformed cell according to claim 7 is used.
The intensity of the external stimulus is quantified using an increase rate of bioluminescence intensity based on expression of a reporter gene after addition of a substrate (RLU / sec) or an area of a response curve as an index, 8. The analysis method according to 8.
The analysis method according to claim 8 or 9, wherein the analysis method is a reporter gene assay method or a two-hybrid assay method.
An analysis method characterized in that, among the ratios of the three types of plasmids constituting the two-hybrid assay method according to claim 10, the ratio of the plasmid for reporter expression is increased more than twice that of the other plasmids.