JP2019010115A - Luciferin-luciferase reaction inhibitor, luciferin-luciferase reaction inhibition method and kit - Google Patents

Abstract

The present invention provides an inhibitor that inhibits the reaction between luciferase and luciferin. A luciferin-luciferase reaction inhibitor, which is a compound having a specific chemical structure or a salt thereof, represented by a compound represented by formula (1-2) or (2-2). [Selection diagram] None

Description

  The present invention relates to a light identification method, a substance detection method, a reporter assay method, a kit, a luciferin-luciferase reaction inhibitor, a luciferin-luciferase reaction inhibition method and apparatus.

Luciferase, which is a photoprotein, is widely used for quantification of gene expression, enzyme activity, ATP, and the like because of its simplicity of measuring luminescence activity and high sensitivity.
An accurate comparative standard (internal standard) is required for accurate luminescence activity measurement. For this purpose, an assay system that can measure a plurality of luminescence activities simultaneously is desirable. There is a dual luciferase assay as a technique that satisfies such a requirement, and the following techniques are known as existing dual luciferase assays.

In the conventional dual luciferase assay, there is a method using two types of luciferases with different substrates (Dual-Luciferase reporter: DLRA, or sold as a dual sea pansy luminescence kit), which is widely used for gene expression quantification and the like. (Non-Patent Document 1). The left side of FIG. 7 is a schematic diagram for explaining the procedure of DLRA.
There is also a method using two types of luciferases having different emission wavelength bands. This is because, for example, green light emission and red light emission can be measured independently by switching the polarizing filter at the time of luminescence measurement using multicolor luciferase. Unlike the DLRA, the same luminescent substrate (D- Luciferin) can be used. This is called a dual-color luciferase assay (DCLA) or a multi-color look reporter assay system, and is commercially available and widely used for gene expression quantification and the like (Non-patent Document 2). The right side of FIG. 7 is a schematic diagram for explaining the procedure of DCLA.

Matsuo N, Minami M, Maeda T, Hiratsuka K, Dual luciferase assay for monitoring gene expression in higher plants., Plant Biotechnology, 18, 71-75, 2001 Ogura R, Matsuo N, Wako N, Tanaka T, Ono S, Hiratsuka K, Multi-color luciferases as reporters for monitoring transient gene expression in higher plants.Plant Biotechnol. 22, 151-155. 2005

The DLRA listed above utilizes, for example, the difference in substrate specificity and reaction conditions between firefly luciferase (Fluc) and Renilla luciferase (Renluca luciferase: Rluc). After measuring the Fluc activity, the Rluc activity is measured by setting the reaction conditions such that Fluc is inactivated simultaneously with the addition of coelenterazine, which is the Rluc substrate. Therefore, in this method, it is necessary to prepare two different types of luminescent substrates and corresponding buffer solutions.
In the DCLA mentioned above, it is possible to measure, for example, green light emission and red light emission independently by switching the polarizing filter at the time of luminescence measurement using multicolor luciferase. Unlike DLRA, the same luminescent substrate is used. (D-luciferin) can be used. However, in DCRA, it is necessary to prepare a measuring instrument equipped with a change filter, and the quantification limit of the luminescence activity is greatly reduced by the intervention of the polarizing filter.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a light identification method that is simpler than the conventional DLRA and can perform a luciferase assay with higher sensitivity than DCLA.

  In order to solve the above problems, the present invention provides a luciferin-luciferase reaction inhibitor, a luciferin-luciferase reaction inhibition method, and a kit having the following characteristics.

［式（１）中、
Ｘ^１はＣＨ又はＮを表し、Ｒ^１はハロゲン原子を表し、Ｒ^２は炭素数１〜４の直鎖状又は分岐鎖状のアルキル基を表す。
ｎはＲ^１の数を表し、０〜３のいずれかの整数であり、ｎが２以上のとき、Ｒ^１同士は互いに同一でも異なっていてもよい。ｍはＲ^２の数を表し、０〜２のいずれかの整数であり、ｍが２のとき、Ｒ^２同士は互いに同一でも異なっていてもよい。］

［式（２）中、
Ｘ^２はＣＨ又はＮを表し、Ｒ^３はハロゲン原子を表し、Ｒ^４は単結合又は２価の連結基を表し、Ｒ^５は炭素数１〜４の直鎖状又は分岐鎖状のアルキル基を表す。
ｐはＲ^３の数を表し、０〜３のいずれかの整数であり、ｐが２以上のとき、Ｒ^３同士は互いに同一でも異なっていてもよい。ｑはＲ^５の数を表し、０〜２のいずれかの整数であり、ｑが２のとき、Ｒ^５同士は互いに同一でも異なっていてもよい。］
＜２＞ ルシフェラーゼとルシフェリンとの反応を阻害する前記＜１＞に記載の阻害剤を用いることを特徴とするルシフェリン−ルシフェラーゼ反応阻害方法。
＜３＞ 以下の工程；
第１ルシフェラーゼ、発光量の測定対象であり発光量測定時に前記第１ルシフェラーゼと反応して光Ａを発する第１発光物質である第１ルシフェリン、及び発光量の測定対象であり発光量測定時に光Ｂを発する第２発光物質（ただし、蛍光物質を除く）を含む測定対象物（ただし、前記第２発光物質がルシフェリンであり、前記測定対象物が、第１ルシフェリン、第１ルシフェラーゼ、前記ルシフェリン、及び前記ルシフェリンと反応させる第２ルシフェラーゼを含むとき、前記第１ルシフェラーゼと前記第２ルシフェラーゼとは異なる。）の、前記第１ルシフェラーゼと前記第１ルシフェリンとを反応させ、前記測定対象物から発せられた光の発光量Ｌ１を測定する第１測定工程、
前記第１ルシフェラーゼと前記第１ルシフェリンとの反応を阻害する阻害剤と前記測定対象物とを接触させて、前記第１ルシフェラーゼと前記ルシフェリンとの反応によって生じる発光量を低下させ、測定対象物から発せられた発光量Ｌ２を測定する第２測定工程、
前記発光量Ｌ１の値と、前記発光量Ｌ２の値と、予め取得された前記阻害剤による前記第１発光物質である第１ルシフェリンの発光量の低下率と、予め取得された前記阻害剤による前記第２発光物質の発光量の低下率と、に基づいて、前記第１ルシフェラーゼと前記第１ルシフェリンの反応により生じた光Ａの発光量、及び前記第２発光物質により生じた光Ｂの発光量を算出し、前記光Ａの発光量と前記光Ｂの発光量とを識別する識別工程、
を含む光識別方法を行うためのキットであって、前記＜１＞に記載の阻害剤を備えることを特徴とするキット。 <1> A luciferin-luciferase reaction inhibitor, which is a compound represented by the following general formula (1), a compound represented by the following general formula (2), or a salt thereof.

[In Formula (1),
X ¹ represents CH or N, R ¹ represents a halogen atom, and R ² represents a linear or branched alkyl group having 1 to 4 carbon atoms.
n represents the number of R ¹ and is an integer of 0 to 3. When n is 2 or more, R ¹ may be the same or different from each other. m represents the number of R ² and is an integer of 0 to 2, and when m is 2, R ² may be the same as or different from each other. ]

[In Formula (2),
X ² represents CH or N, R ³ represents a halogen atom, R ⁴ represents a single bond or a divalent linking group, and R ⁵ represents a linear or branched alkyl group having 1 to 4 carbon atoms. Represents.
p represents the number of R ³ and is an integer of 0 to 3, and when p is 2 or more, R ³ may be the same or different from each other. q represents the number of R ⁵ , and is an integer of 0 to 2, and when q is 2, R ⁵ may be the same as or different from each other. ]
<2> A method for inhibiting luciferin-luciferase reaction, comprising using the inhibitor according to the above <1> which inhibits the reaction between luciferase and luciferin.
<3> The following steps;
The first luciferase, the first luciferin, which is a measurement target of the luminescence level and reacts with the first luciferase to emit light A when measuring the luminescence level, and the first luciferin, which is the measurement target of the luminescence level, and light when measuring the luminescence level. A measurement object including a second luminescent substance that emits B (excluding a fluorescent substance) (provided that the second luminescent substance is luciferin, and the measurement object is a first luciferin, a first luciferase, the luciferin, And the second luciferase to be reacted with the luciferin is different from the first luciferase and the second luciferase), the first luciferase and the first luciferin are reacted, and emitted from the measurement object. A first measurement step of measuring the emitted light amount L1
An inhibitor that inhibits the reaction between the first luciferase and the first luciferin is brought into contact with the measurement object, and the amount of luminescence generated by the reaction between the first luciferase and the luciferin is reduced. A second measuring step for measuring the emitted light amount L2,
The value of the light emission amount L1, the value of the light emission amount L2, the rate of decrease in the light emission amount of the first luciferin that is the first light emitting substance by the inhibitor acquired in advance, and the inhibitor acquired in advance. The light emission amount of light A generated by the reaction of the first luciferase and the first luciferin based on the decrease rate of the light emission amount of the second light emitting material, and the light emission of light B generated by the second light emitting material A discriminating step for calculating the amount and identifying the light emission amount of the light A and the light emission amount of the light B;
A kit for performing a light identification method comprising: the inhibitor according to <1>.

  The present invention also relates to the following light identification method, reporter assay method, kit, and apparatus.

(1) The following steps;
The first luciferase, the first luciferin, which is a measurement target of the luminescence level and reacts with the first luciferase to emit light A when measuring the luminescence level, and the first luciferin, which is the measurement target of the luminescence level, and light when measuring the luminescence level. A measurement object including a second luminescent substance that emits B (excluding a fluorescent substance) (provided that the second luminescent substance is luciferin, and the measurement object is a first luciferin, a first luciferase, the luciferin, And the second luciferase to be reacted with the luciferin is different from the first luciferase and the second luciferase), the first luciferase and the first luciferin are reacted, and emitted from the measurement object. A first measurement step of measuring the emitted light amount L1
An inhibitor that inhibits the reaction between the first luciferase and the first luciferin is brought into contact with the measurement object, and the amount of luminescence generated by the reaction between the first luciferase and the luciferin is reduced. A second measuring step for measuring the emitted light amount L2,
The value of the light emission amount L1, the value of the light emission amount L2, the rate of decrease in the light emission amount of the first luciferin that is the first light emitting substance by the inhibitor acquired in advance, and the inhibitor acquired in advance. The light emission amount of light A generated by the reaction of the first luciferase and the first luciferin based on the decrease rate of the light emission amount of the second light emitting material, and the light emission of light B generated by the second light emitting material A discriminating step for calculating the amount and identifying the light emission amount of the light A and the light emission amount of the light B;
An optical identification method comprising:
(2) The light according to (1), wherein the second luminescent substance is luciferin, and the measurement object includes a first luciferin, a first luciferase, the luciferin, and a second luciferase that is reacted with the luciferin. Identification method.
(3) The light identification method according to (2), wherein the luciferin is the same substance as the first luciferin.
(4) The amount of light A generated by the reaction of the first luciferase and the first luciferin in the absence of the inhibitor is the amount of light A1,
The amount of light A generated by the reaction of the first luciferase and the first luciferin in the presence of the inhibitor is defined as a light emission amount A2.
The amount of light B generated by the reaction between the second luciferase and the luciferin in the absence of the inhibitor is the amount of light B1,
When the light emission amount of light B generated by the reaction between the second luciferase and the luciferin in the presence of the inhibitor is the light emission amount B2,
The ratio of the decrease rate of the light emission amount of light A (A2 / A1) and the decrease rate of the light emission amount of light B (B2 / B1) is 1.1 times or more, as described in (2) or (3) above Light identification method.
(5) The above-mentioned (1) to (4), wherein the first luciferin is D-luciferin or a D-luciferin analog that is a compound capable of producing light by reacting with luciferase using D-luciferin as a substrate. The light identification method according to any one of the above.
(6) The optical identification method according to any one of (1) to (5), wherein the first luciferase is Fluc, Luc2, SLO, SLG, or CBR.
(7) The optical identification according to any one of (1) to (6), wherein the measurement object is a cell, and the light emitted from the cell of the measurement object is non-destructively measured. Method.
(8) A reporter assay method using the light identification method according to any one of (1) to (7).
(9) A fusion gene in which the first luciferase, the first luciferin that is the first luminescent substance, and the measurement object containing the second luminescent substance are linked to the first luciferase gene downstream of the gene regulatory region is introduced. The reporter assay method according to (8) above, comprising a recombinant cell expressing the first luciferase or an extract thereof.
(10) A kit for performing the light identification method according to any one of (1) to (7) or the reporter assay method according to (8) or (9),
An inhibitor that inhibits a reaction between the first luciferase and the first luciferin;
An instruction manual explaining the contents for performing the light identification method according to any one of (1) to (7) or the reporter assay method according to (8) or (9);
A kit comprising:
(11) An apparatus for performing the light identification method according to any one of (1) to (7), wherein (a) a measurement for measuring a light emission amount of light emitted from the measurement object. And (b) a reaction between the first luciferase and the first luciferin based on the value of the luminescence L1 measured by the measurement means and the value of the luminescence L2 measured by the measurement means. And a calculating means for identifying the light A generated by the light and the light B generated by the second luminescent material.

  According to the present invention, a luciferin-luciferase reaction inhibitor having luciferase inhibitory activity can be provided, and can be used in the above-described light identification method and the above-described kit.

It is a schematic diagram for demonstrating the procedure of the optical identification method in this embodiment. It is a schematic diagram for demonstrating the procedure of the modification of the light identification method in this embodiment. It is a schematic diagram which shows an example of a structure of the apparatus which concerns on this invention. It is a graph which shows the result of inhibition by the inhibitor of the luciferin-luciferase reaction in an Example. It is a graph which shows the result of inhibition by the inhibitor of the luciferin-luciferase reaction in an Example. In an Example, it is a graph which shows the result of having calculated the value of the promoter activity normalized by the internal standard by discriminating light emission by two types of luciferin-luciferase reactions in spinach cells expressing two types of luciferases. It is a schematic diagram for demonstrating the procedure of the luciferase assay using two types of conventional luciferases.

<Light identification method>
The optical identification method of the present invention
The following steps:
The first luciferase, the first luciferin that is the first luminescent substance, and the measurement object that includes the second luminescent substance are reacted with the first luciferase and the first luciferin, and the light emitted from the measurement object A first measurement step for measuring the light emission amount L1,
An inhibitor that inhibits the reaction between the first luciferase and the first luciferin is brought into contact with the measurement object, and the amount of luminescence generated by the reaction between the first luciferase and the luciferin is reduced. A second measuring step for measuring the emitted light amount L2,
Based on the value of the light emission amount L1 and the value of the light emission amount L2, the light A generated by the reaction of the first luciferase and the first luciferin and the light B generated by the second light emitting substance are distinguished. An identification step.

(First measurement process)
The light identification method of the present embodiment includes a first luciferase, a first luciferin that is a first luminescent material, a luciferin that is a second luminescent material, and a first luciferase that is reacted with the luciferin. A first measuring step of reacting luciferase with the first luciferin and measuring a light emission amount L1 of light emitted from the measurement object;

FIG. 1 is a diagram schematically showing a procedure of a light identification method in the first embodiment according to the present invention. The measurement object of the present embodiment includes Fluc as the first luciferase that is the first luminescent substance, D-luciferin as the first luciferin, and CBG (Click Beetle Green) as the second luciferase. In the present embodiment, the luciferin and the first luciferin are the same substance, and both Fluc (first luciferase) and CBG (second luciferase) react with D-luciferin to emit light by the luciferin-luciferase reaction. Occurs. D-luciferin reacted with Fluc is the first luminescent material, and D-luciferin reacted with CBG is the second luminescent material.
In the present embodiment, the measurement object is a cell extract preparation. The cell from which the cell extract is derived contains Fluc and CBG expressed in the cell. The said cell extract, D-luciferin, and a buffer are mixed, and the measuring object of the 1st measurement process which is a liquid containing Fluc, CBG, and D-luciferin is obtained. The measurement object in the first measurement step is a liquid that does not contain an inhibitor described later, and the measurement of the light emission amount L1 is a measurement in the absence of the inhibitor.

  Next, the light emission amount L1 of light emitted from the measurement object in the first measurement step is measured. The amount of luminescence can be measured using a known luminometer. In the measurement object in the first measurement step, light A is generated by the reaction of Fluc and D-luciferin, and light B is generated by the reaction of CBG and D-luciferin. The amount of light A generated by the reaction between the first luciferase and the first luciferin in the absence of an inhibitor is defined as the amount of luminescence A1, and the reaction between the second luciferase and the first luciferin in the absence of an inhibitor. Assuming that the light emission amount of the light B generated by the above is the light emission amount B1, the value of the light emission amount L1 of the light emitted from the measurement object in the first measurement step is the light emission amount L1 = the light emission amount A1 + the light emission amount B1.

(Second measurement process)
The light identification method of the present embodiment is a method in which an inhibitor that inhibits the reaction between the first luciferase and the first luciferin is brought into contact with the measurement object, and light emission generated by the reaction between the first luciferase and the luciferin. A second measurement step of measuring the light emission amount L2 emitted from the measurement object by reducing the amount.

  As shown in FIG. 1, in this embodiment, an inhibitor that inhibits the reaction between the first luciferase and the first luciferin is added to the liquid that is the measurement object in the first measurement step, and the second measurement step. The measurement object is obtained. The measurement object in the second measurement step is a liquid containing an inhibitor, and the measurement of the light emission amount L2 is a measurement in the presence of the inhibitor.

Next, the light emission amount L2 of the light emitted from the measurement object in the second measurement step is measured. The amount of luminescence can be measured using a known luminometer.
The amount of light A generated by the reaction of the first luciferase and the first luciferin in the presence of an inhibitor is defined as the amount of light emission A2, and is generated by the reaction of the second luciferase and the first luciferin in the presence of an inhibitor. Assuming that the light emission amount of the light B is the light emission amount B2, the value of the light emission amount L2 of the light emitted from the measurement object in the second measurement step is the light emission amount L2 = the light emission amount A2 + the light emission amount B2.
Since the inhibitor inhibits the reaction between the first luciferase and the first luciferin, the light emission amount L2 emitted from the measurement object in the second measurement step is emitted from the measurement object in the first measurement process. It is lower than the light emission amount L1 of the emitted light. In the present embodiment, an inhibitor that inhibits the reaction between Fluc and D-luciferin is used as the inhibitor, and the inhibitor has an effect of reducing the value of luminescence A2 to 1/10 of A1 (FIG. 1).

In addition, in this embodiment, the inhibitor was added to the measurement object of the 1st measurement process used for the 1st measurement process, and the measurement object of the 2nd measurement process was obtained, but the measurement of a 2nd measurement process The element of the measurement object of the first measurement process included in the object does not necessarily have to have undergone the first measurement process.
For example, two liquids, that is, the liquid of the measurement object of the first measurement process measured in the first measurement process and the liquid that is the composition of the measurement object of the first measurement process and is not measured in the first measurement process are previously stored. An inhibitor may be added to a liquid that is not measured in the first measurement step and used as a measurement object in the second measurement step.

  In addition, in this embodiment, since the inhibitor was added to the measurement object of the 1st measurement process used for the 1st measurement process, and the measurement object of the 2nd measurement process was obtained, after the 1st measurement process, The 2nd measurement process was performed. However, it is not always necessary to perform the second measurement process after the first measurement process, and the second measurement process may be performed before the first measurement process, and the second measurement process is performed simultaneously with the first measurement process. You may do it.

(Identification process)
The light identification method according to the present embodiment includes the light A generated by the reaction of the first luciferase and the first luciferin based on the value of the light emission amount L1 and the value of the light emission amount L2, and the second luciferase. And identifying the light B generated by the reaction of the first luciferin.

  Here, the light is identified based on the value of the light emission amount L1 and the value of the light emission amount L2 even when the light A and the light B are detected without being distinguished from the measurement object. The light A and the light B are discriminated by calculating the light emission amount derived from the light A and the light emission amount derived from the light B among the detected light emission amounts.

In order to calculate the light emission amount derived from the light A and the light emission amount derived from the light B, the reduction rate (A2 / A1) of the light emission amount of the light A by the inhibitor needs to be known in advance.
In order to obtain the reduction rate of the light emission amount of the light A, the first measurement step and the second measurement step may be performed under the same conditions except that the second light emitting material is not included. In this case, since the light emitted from the measurement object is derived only from the light A, the light emission amount A1 in the absence of the inhibitor and the light emission amount A2 in the presence of the inhibitor are measured to reduce the light emission amount. What is necessary is just to obtain | require a rate (A2 / A1).

In the present embodiment, the inhibitor A inhibits only the reaction between the first luciferase and the first luciferin, and does not inhibit the reaction between the second luciferase and the first luciferin. However, the inhibitor used in the present invention may be one that changes not only the reaction between the first luciferase and the first luciferin but also the amount of luminescence of the second luminescent substance. In addition to the reaction with 1 luciferin, the reaction between the second luciferase and the first luciferin may be inhibited.
Also in this case, it is necessary that the rate of change of the light emission amount of the second luminescent substance by the inhibitor is known in advance. For example, when the second luminescent substance is the first luciferin that reacts with the second luciferase and the inhibitor inhibits the reaction between the second luciferase and the first luciferin, the decrease rate of the light B emission amount (B2 / B1 ) Must be known in advance.
In order to obtain the reduction rate of the light emission amount of light B, the first measurement step and the second measurement step may be performed under the same conditions except that the first luminescent substance and the first luciferin are not included. In this case, since the light emitted from the measurement object is derived only from the light B, the light emission amount B1 in the absence of the inhibitor and the light emission amount B2 in the presence of the inhibitor are measured to reduce the light emission amount. What is necessary is just to obtain | require a rate (B2 / B1).

  Assuming that the decrease rate of the light emission amount of light A (A2 / A1) is x% and the decrease rate of the light emission amount of light B (B2 / B1) is y%, as shown by the following formula (I): , L1 = A1 + B1, L2 = x / 100A1 + y / 100B1, and A1 and B1 can be calculated.

  The difference in the decrease rate of the light A emission amount (A2 / A1) and the decrease rate of the light B emission amount (B2 / B1) by the inhibitor reflects the specificity of the inhibitor. Since the reduction rate of the luminescence amount is almost constant under the same reaction conditions, if the information about the reduction rate is known, the reduction rate is obtained again when the light identification method of the present invention is performed. There is no need.

  It is preferable that the ratio value between the decrease rate of the light emission amount of light A (A2 / A1) and the decrease rate of the light emission amount of light B (B2 / B1) is larger. This can reduce the possibility that the effect of the inhibitor falls within the error range, and can further improve the accuracy of light separation. The ratio of the decrease rate of the emission amount of light A (A2 / A1) to the decrease rate of the emission amount of light B (B2 / B1) is 1.1 times or more, 1.5 times or more, 2 times or more, 2 .5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 5.5 times or more, 6 times or more, 6.5 times or more is preferable, 7 times or more 7.5 times or more, 8 times or more, 8.5 times or more are more preferable, 9 times or more, 9.5 times or more, 10 times or more, 15 times or more, or 20 times or more are more preferable.

  In addition, you may perform the optical identification method of this invention by making the said light-emission amount into light emission intensity. The amount of luminescence may be measured as RLU (relative light unit).

(Luminescent material)
Luciferase is a generic term for enzymes that catalyze bioluminescence, and luciferin is a generic term for compounds that react with luciferase to produce light.
Various types of luciferases and luciferins have been discovered so far, and representative luciferases include Renilla luciferase (Rluc), firefly luciferase (Fluc), green light-emitting protein (CBG) derived from Click beetle. Examples thereof include Click Belt Green and Red Photoprotein (CBR). Representative luciferins include Renilla luciferin, firefly luciferin, coelenterazine, and the like.
Luciferase has substrate properties. The luciferin that reacts with a specific luciferase to produce light can be appropriately selected by those skilled in the art.
The luciferase may be a naturally occurring protein that exists in nature, or may be a mutant or artificially modified protein having an amino acid sequence, modification, addition, or the like that is different from the naturally occurring protein. The luciferin may be a naturally occurring natural compound or a compound having a structure different from that of the natural type as long as it reacts with luciferase to generate light.

  In the present specification, the first luciferase is a luciferase in which the reaction between luciferase and luciferin is inhibited by an inhibitor. In the present specification, the first luciferin that is the first luminescent substance is a compound that reacts with the first luciferase to generate light. The first luciferin is preferably D-luciferin or an analog thereof. The analog of D-luciferin is preferably a compound that reacts with luciferase using D-luciferin as a substrate to produce light. Examples of the analog of D-luciferin include Aka Lumine (registered trademark). The first luciferase is preferably luciferase using D-luciferin or an analog thereof as a substrate.

  The light A generated by the reaction between the first luciferase and the first luciferin is not limited to the light itself generated from the first luciferin. Similarly, the light B generated by the second light emitting material is not limited to the light itself generated from the second light emitting material. For example, the light A may be generated by bioluminescence resonance energy transfer (BRET), and the light generated by the reaction between the first luciferase and the first luciferin undergoes energy transfer. The resulting light is also included.

  The second light emitting substance is not particularly limited as long as it is a substance that emits light. Examples of the substance used as the second luminescent substance include fluorescent dyes such as fluorescein, rhodamine and cyanine, fluorescent proteins such as green fluorescent protein (GFP), luminescent proteins such as aequorin, luciferin and the like.

When the second luminescent substance used in the light identification method of the present invention is luciferin, and the measurement object includes the first luciferin, the first luciferase, the luciferin, and the second luciferase that reacts with the luciferin, As described in one embodiment, the luciferin is preferably the same substance as the first luciferin.
In the conventional DLRA, for example, firefly luciferase (Fluc) and Renilla luciferase (Rluc) are used and the difference in substrate specificity and reaction conditions is utilized, so two different luminescent substrates are used. It is necessary to prepare a buffer solution corresponding to them.
On the other hand, in the light identification method of the present embodiment, by selecting an appropriate luciferase and luciferin, it is possible to cause both the first luciferase and the second luciferase to react with the first luciferin, so the reaction conditions for luciferase are changed. Without doing so, the reaction of both the first luciferase and the first luciferin and the second luciferase and the first luciferin can be carried out in the same reaction system. Therefore, it is not necessary to change the buffer solution, and the luciferase assay can be performed more easily.
Various combinations can be exemplified as the combination of the first luciferase, the second luciferase and the luciferin that react with the common luciferin. Examples of the luciferase that reacts with D-luciferin include, for example, Fluc, CBR, CBG, and SLG (green light emission). Different ones may be selected as the first luciferase and the second luciferase from the group consisting of luciferase), SLO (orange luminescence luciferase), and SLR (red luminescence luciferase), respectively.

(Measurement object)
The measurement object according to the light identification method of the present invention is not particularly limited as long as it includes the first luciferase, the first luciferin that is the first luminescent substance, and the second luminescent substance. Or the substance derived from a cell is mentioned. Making a cell a measurement object includes making a measurement object a cell population, a tissue, an organ, or an individual organism that is a collection of cells. Examples of cells include eukaryotic cells such as plant cells, plant bodies, animal cells, insect cells, yeasts, and fungi, and include unicellular organisms and microorganisms. Examples of cell-derived substances include cell extracts, cell culture supernatants, and preparations thereof.
The measurement object does not need to uniformly contain the first luciferase, the first luciferin that is the first luminescent substance, and the second luminescent substance. For example, when the measurement target is an individual, the first luciferase, the first luciferin that is the first luminescent substance, and the second luminescent substance may be included only in some of the cells that constitute the individual. As another example, the first luciferase and the first luciferin that is the first luminescent substance are included only in some of the cells constituting the individual, and the second luminescent substance is included in a part different from the above part. It may be.

In the light identification method of the present invention, the measurement object may be a cell, and the light emitted from the cell of the measurement object may be measured non-destructively.
In the DLRA listed above, for example, firefly luciferase (Fluc) and Renilla luciferase (Rluc) are used, firefly luciferin is used as a substrate for Fluc, and coelenterazine may be used as a substrate for Rluc. . However, since coelenterazine is a relatively high polymer, it does not easily penetrate into cells. Therefore, in order to react Rluc and coelenterazine expressed in cells, the measurement object must be a cell extract obtained by destroying cells.
On the other hand, in the optical identification method of the present invention, as described above, the first luciferase and the second luciferase can be used to construct a reaction system using the same type of luciferin. The need to do can be reduced.
For example, in the first embodiment, the preparation of the cell extract is the measurement object. However, in the first embodiment, only firefly luciferin having excellent cell permeability is used as the luminescent substrate. It is also possible to measure non-destructively the light generated from the cells of the measurement object. In addition, the luciferin-luciferase reaction inhibitor of the present invention, which will be described later, is excellent in cell permeability, so that light generated from the cells of the measurement target can be measured non-destructively.

<Method for detecting substances>
The method for detecting a substance of the present invention uses the optical identification method of the present invention. Detection includes measurement and quantification. The light identification method of the present invention can be widely used for a method for detecting a substance conventionally performed by using a luciferin-luciferase reaction.
For example, since the luminescence reaction between firefly luciferase and firefly luciferin is a reaction that occurs only in the presence of ATP, the presence of ATP in the measurement object can be detected by using the light discrimination method of the present invention. When the detection target is ATP, as one embodiment of the method for detecting a substance of the present invention,
The following steps:
The first luciferase and the first luciferin are reacted with a test sample that may contain ATP as a detection target, a first luciferase, a first luciferin as a first luminescent substance, and a measurement target including a second luminescent substance. A first measurement step of measuring a light emission amount L1 of light emitted from the measurement object,
An inhibitor that inhibits the reaction between the first luciferase and the first luciferin is brought into contact with the measurement object, and the amount of luminescence generated by the reaction between the first luciferase and the luciferin is reduced. A second measuring step for measuring the emitted light amount L2,
Based on the value of the light emission amount L1 and the value of the light emission amount L2, the light A generated by the reaction of the first luciferase and the first luciferin and the light B generated by the second light emitting substance are distinguished. Identification process,
Can be included.
Similarly to ATP, enzyme activity such as ATPase, measurement of cells producing ATP, measurement of microorganisms, and the like can be performed.

  In the method for detecting a substance of the present invention, for example, the first luminescent substance or the second luminescent substance may be used as a standard substance (control), so that the substance can be detected simply and with high accuracy.

In addition to the ATP listed above, the presence of an element essential for the luciferin-luciferase reaction in the reaction system can be detected and detected. Elements essential for the luciferin-luciferase reaction include ATP, luciferin, luciferase, Mg ²⁺ , and oxygen.

  The substance to be measured may be detected using the fact that the above-mentioned elements essential for the luciferin-luciferase reaction are generated in the reaction system. For example, if a reaction system in which a luciferin serving as a luciferase substrate is produced by a reaction catalyzed by the enzyme to be detected is constructed in the measurement object, the presence of the enzyme can be detected.

The light identification method of the present invention can be used in combination with other luciferase assay methods. For example, in addition to the optical identification method of the present invention, performing DLRA can be exemplified. For example, the measurement object contains a third luciferase that uses a luciferin that is different from the first luciferase to be reacted in the light identification method of the present invention, and the measurement object subjected to the light identification method of the present invention includes A reaction solution for 3 luciferase and luciferin to be reacted with the third luciferase are added, and the third luciferase and luciferin are reacted.
FIG. 2 illustrates the case where Rluc is used as the third luciferase. In this way, the types of light that can be identified and detected can be increased.

<Reporter assay method>
The reporter assay method of the present invention uses the optical identification method of the present invention. Reporter assay is a technique for detecting gene expression, gene regulation, cell response, and the like via a signal from a reporter. The light identification method of the present invention can be widely used in reporter assays that have been conventionally performed using a luciferin-luciferase reaction.
For example, the measurement object in the optical identification method of the present invention is a recombinant cell in which a fusion gene in which the first luciferase gene is linked is introduced downstream of a gene regulatory region and the first luciferase is expressed, or extraction thereof It may contain things. Here, the gene regulatory region may be any sequence that can control transcription of a gene linked downstream, and includes, for example, a promoter, an enhancer, a hormone response sequence, and the like.
The luciferase gene has a naturally-occurring natural-type base sequence, but has a mutant-type or artificially-modified base sequence having a base acid sequence, modification, addition, etc. different from the natural type. It may be. For example, Luc2 has a codon optimized for the purpose of increasing the expression efficiency of firefly luciferase in mammalian cells. As for luciferin, it may be a naturally occurring natural compound or a compound having a structure different from that of the natural type as long as it reacts with luciferase to generate light.

  The cell to be a recombinant cell is not particularly limited and is preferably a eukaryotic cell such as a plant cell, an animal cell, an insect cell, yeast, or a fungus, and more preferably a plant cell. When obtaining transformants of plant cells, the cells into which the fusion gene is introduced can include all types of plant cells that can be regenerated into plants. Examples thereof include cultured cells, protoplasts, shoot primordia, multiblasts, hairy roots, and callus. The plant species to be transformed is not particularly limited, and may be a monocotyledonous plant or a dicotyledonous plant, but is preferably an agricultural crop or a horticultural crop. For example, grains such as rice, barley, wheat, rye, corn, sugar cane, crops that form rhizomes or tuberous roots such as potatoes, sweet potatoes, legumes such as kidney beans, broad beans, peas, peanuts, sesame, rapeseed, cottonseed, Preferred are seed crops such as sunflower and safflower, crops having fruits such as apples, melons and grapes, crops such as tomatoes and eggplants, and various flower plants. Plant transformants can convert cells into plants by carrying out a predetermined regeneration step. The method of regeneration varies depending on the type of plant, but various known methods can be used.

  Recombinant cells include, without limitation, cells of all types and forms. In addition to various cells, animals and plants, eukaryotic cells that can constitute the animals and plants, and tissues and organs that are a part thereof. As well as germ cells. It also includes viral particles. Furthermore, the propagation medium (seed, rhizome, fruit, cutting ear, etc.) which is a part of plant individual is also included.

  The detection target includes a second luminescent material. The second light-emitting substance is not particularly limited as long as it is a substance that emits light, and those described in the above <light identification method> can be similarly used.

In the reporter assay method of the present invention, for example, a fusion gene in which the first luciferase gene is linked downstream of the gene regulatory region is introduced into the measurement object, and further, a reporter gene is linked downstream of the gene regulatory region. Or a recombinant cell expressing the first luciferase and the reporter gene, or an extract thereof.
Examples of the reporter gene include fluorescent proteins such as GFP and luciferase, and luciferase is preferable. Examples of the luciferase include those described above.
Therefore, the detection target is introduced with a fusion gene in which the first luciferase gene is linked downstream of the gene regulatory region, and is further introduced with a fusion gene in which the second luciferase gene is linked downstream of the gene regulatory region. And may contain a recombinant cell expressing the first luciferase and the second luciferase or an extract thereof. Examples of the gene regulatory region include those described above. An example of an embodiment of the reporter assay method of the present invention is shown below.

First, a gene regulatory region of a target gene whose gene expression level is to be measured is linked upstream of the first luciferase gene to obtain a first luciferase fusion gene. The first luciferase fusion gene may be constructed as an expression vector capable of expressing the gene in the cell of the measurement target. Similarly, the gene regulatory region of the gene to be used as an internal control is linked upstream of the second luciferase gene to obtain a second luciferase fusion gene. The second luciferase fusion gene may be constructed as an expression vector capable of expressing the gene in the cell of the measurement target.
The expression vector may be constructed as a chromosome integration type vector in addition to those capable of autonomous replication outside the chromosome. The form may be a linear fragment, plasmid, cosmid, phage, virus, transposon, yeast artificial chromosome (YAC), plant artificial chromosome (PAC), mammalian artificial chromosome (MAC), or the like. Among these, a plasmid vector is preferable.

The first luciferase fusion gene and the second luciferase fusion gene are introduced into the cells.
The method for introducing the fusion gene into cells may be performed by a conventional method, and examples thereof include physical means such as electroporation and gene gun, and biological means such as Agrobacterium method. If necessary, the cells are incubated to express the first luciferase and the second luciferase.
Next, the cells are immersed in a solution containing luciferin that reacts with the first luciferase and the second luciferase to introduce luciferin into the cells. In this way, it is only necessary to obtain a cell that is a measurement object including the first luciferase, the first luciferin that is the first luminescent substance, the luciferin that is the second luminescent substance, and the second luciferase that is reacted with the luciferin.
Next, the first measurement step, the second measurement step, and the identification step according to the light identification method of the present invention are performed to identify the light A generated by the reaction of the first luciferin and the light B generated by the second luciferin. Light A represents gene expression of the target gene, and light B represents gene expression of the internal control gene.

  In the above example, the target of detection of the reporter assay is the expression level of the target gene, and the case where the first luciferase gene is linked downstream of the target gene regulatory region is exemplified. Cell information such as cell response and signal transduction can be obtained by using a gene regulatory region of a factor relating to various cell responses or signal transduction instead of the regulatory region. Examples of cell information include immune response, apoptosis, cytotoxicity, cell proliferation, cell differentiation, cell cycle and the like.

  In the reporter assay method for a substance of the present invention, for example, the first luminescent substance or the second luminescent substance may be used as a standard substance (control), so that the substance can be detected simply and with high accuracy.

The reporter assay method of the present invention can be used in combination with other luciferase assay methods. For example, in addition to the reporter assay method of the present invention using the optical identification method of the present invention, performing DLRA can be exemplified. For example, the measurement object contains a third luciferase that uses a luciferin that is different from the first luciferase to be reacted in the light identification method of the present invention, and the measurement object subjected to the light identification method of the present invention includes A reaction solution for 3 luciferase and luciferin to be reacted with the third luciferase are added, and the third luciferase and luciferin are reacted.
FIG. 2 illustrates the case where Rluc is used as the third luciferase. In this way, the types of light that can be identified and detected can be increased.

(Screening method)
Furthermore, by using the reporter assay method of the present invention, it is possible to evaluate the effect of a compound on cells and to screen for physiologically active substances, drugs and the like.
Hereinafter, a case where screening of a physiologically active substance related to an immune response is performed will be described.

For example, first, a fusion gene in which the first luciferase gene is linked downstream of the gene regulatory region of the immune response-related factor is introduced into the cell, and further, the second luciferase is further downstream of the gene regulatory region of the gene used as an internal control. A recombinant cell expressing a first luciferase and a second luciferase is prepared by introducing a fusion gene linked with a gene. Subsequently, the test substance suspected of being a physiologically active substance is brought into contact with the recombinant cell, and luciferin is brought into contact with the recombinant cell. There is no restriction | limiting in particular as a test substance, For example, a peptide, protein, a non-peptide compound, a low molecular compound, a synthetic compound, etc. are mentioned. The contact between the test substance and the cell may be a contact between the test sample containing the test substance and the cell. There is no restriction | limiting in particular as a test sample, For example, it is a fermentation product, a cell extract, a plant extract, an animal tissue extract etc. When the test substance is a physiologically active substance related to immune response, expression of the first luciferase is induced in the cell, and luciferin reacts with the first luciferase and the second luciferase.
Next, the first measurement step, the second measurement step, and the identification step according to the light identification method of the present invention are performed to identify the light A generated by the reaction of the first luciferin and the light B generated by the second luciferin. Light A represents gene expression of an immune response-related factor, and light B represents gene expression of an internal control gene. For example, the same assay is performed in the absence of the test substance in advance to obtain the light emission amounts of light A and light B, and light emission of light A and light B in the presence and absence of the test substance. The test substance may be selected by determining whether the test substance is a physiologically active substance related to an immune response based on the change in the amount. For example, the light emission amount of light A after correction by the light emission amount of light B is increased as compared with the light emission amount of light A after correction by the light emission amount of light B when the test substance is not contacted with a recombinant cell. The test substance may be selected as a candidate for a physiologically active substance related to immune response.

<Kit>
The kit of the present invention is a kit for carrying out the light identification method of the present invention, the detection method of the substance of the present invention, or the reporter assay method of the present invention, wherein the reaction between the first luciferase and the first luciferin It is provided with an inhibitor that inhibits.
The inhibitor is not particularly limited as long as it inhibits the reaction between the first luciferase and the first luciferin according to the light identification method of the present invention, and the inhibitor described in the above <light identification method> is preferable. It can be illustrated.
The kit of the present invention may include a buffer and reagents for luciferin-luciferase reaction, a reaction container, and an instruction manual in addition to the inhibitor.
The buffer has a role of maintaining the pH of the liquid that becomes the reaction system of the luciferin-luciferase reaction at a pH suitable for the luciferin-luciferase reaction. The type of the buffer can be appropriately selected by those skilled in the art according to the type of luciferin to be reacted, the type of luciferase, and the type of the measurement object. The buffer may contain a reagent such as luciferin in advance.

The kit of the present invention may comprise a fusion gene in which the first luciferase gene is linked downstream of the gene regulatory region, and a fusion gene in which the first luciferase gene is linked downstream of the gene regulatory region, and a gene A fusion gene in which the second luciferase gene is linked downstream of the regulatory region may be provided. The fusion gene may be constructed as an expression vector capable of expressing the gene in the cell of the measurement target.
Alternatively, the kit of the present invention may comprise a vector for constructing a fusion gene in which the first luciferase gene is linked downstream of the gene regulatory region of the gene to be detected, and gene regulation of the gene to be detected A vector for constructing a fusion gene in which the first luciferase gene is linked downstream of the region, and a fusion gene in which the second luciferase gene is linked downstream of the gene regulatory region of the gene to be detected A vector may be provided. An example of a vector for constructing a fusion gene in which the first luciferase gene is linked downstream of a gene regulatory region includes a first luciferase gene that encodes a first luciferase protein. By inserting a gene regulatory region of a gene to be detected upstream, a fusion gene can be constructed.
In addition, the kit of the present invention may further include reagents used for obtaining cell information such as immune response, apoptosis, cytotoxicity, cell proliferation, cell differentiation, cell cycle and the like.

<Luciferin-luciferase reaction inhibitor>
The luciferin-luciferase reaction inhibitor of the present invention is a compound represented by the following general formula (1) or a salt thereof, or a compound represented by the following general formula (2) or a salt thereof.

[In Formula (1),
X ¹ represents CH or N, R ¹ represents a halogen atom, and R ² represents a linear or branched alkyl group having 1 to 4 carbon atoms.
n represents the number of R ¹ and is an integer of 0 to 3. When n is 2 or more, R ¹ may be the same or different from each other. m represents the number of R ² and is an integer of 0 to 2, and when m is 2, R ² may be the same as or different from each other. ]

The halogen atom of R ¹ is an element belonging to Group 17 in the periodic table such as F, Cl, Br, and I.
Examples of the linear or branched alkyl group having 1 to 4 carbon atoms of R ² include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert. -A butyl group can be illustrated. The linear or branched alkyl group having 1 to 4 carbon atoms of R ² preferably has 1 to 3 carbon atoms, and more preferably has 1 or 2 carbon atoms.

[In Formula (2),
X ² represents CH or N, R ³ represents a halogen atom, R ⁴ represents a single bond or a divalent linking group, and R ⁵ represents a linear or branched alkyl group having 1 to 4 carbon atoms. Represents.
p represents the number of R ³ and is an integer of 0 to 3, and when p is 2 or more, R ³ may be the same or different from each other. q represents the number of R ⁵ , and is an integer of 0 to 2, and when q is 2, R ⁵ may be the same as or different from each other. ]

The halogen atom of R ³ is an element belonging to Group 17 in the periodic table such as F, Cl, Br, and I.
Examples of the divalent linking group for R ⁴ include an alkylene group, a divalent linking group containing NH, or a combination thereof. When R < ⁴ > is an alkylene group, C1-C3 is preferable and C1-C2 is more preferable.
When R ⁴ is a divalent linking group containing NH, examples of the divalent linking group containing NH include —NH— and —C—NH—.
Examples of the linear or branched alkyl group having 1 to 4 carbon atoms of R ⁵ include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert. -A butyl group can be illustrated. The linear or branched alkyl group having 1 to 4 carbon atoms of R ⁵ preferably has 1 to 3 carbon atoms, and more preferably 1 or 2 carbon atoms.

In the compound represented by the general formula (1), when X ¹ is N, a compound represented by the following general formula (1-1) is exemplified.

[Wherein, R ¹ and R ² are the same as those in the general formula (1). ]

When R ¹ is Cl and R ² is a methyl group, examples of the compound represented by the general formula (1-1) include a compound represented by the following formula (1-2).

In the compound represented by the general formula (2), when X ² is CH, a compound represented by the following general formula (2-1) is exemplified.

[Wherein, R ³ , R ⁴ and R ⁵ are the same as those in the general formula (2). ]

When R ³ is Cl, R ⁴ is —C—, and R ⁵ is a methyl group, the compound represented by the general formula (2-1) is represented by the following formula (2-2). The compound which is made is mentioned.

The compound represented by the general formula (1) can inhibit the reaction between luciferase and luciferin, and as an inhibitor that inhibits the reaction between the first luciferase and the first luciferin in the light identification method of the present invention. Can be preferably used.
As said 1st luciferin which the compound represented by the said General formula (1) inhibits reaction, it is preferable that it is D-luciferin or its analog.

  In the light identification method of the present invention, the second luminescent substance is luciferin, and the measurement object includes a first luciferin, a first luciferase, the luciferin, and a second luciferase that is reacted with the luciferin, and the luciferin is When the first luciferin is the same substance as the first luciferin, the first luciferin that inhibits the reaction of the compound represented by the general formula (1) is preferably D-luciferin or an analog thereof. At this time, from the viewpoint of increasing the ratio between the rate of decrease in the light emission amount of light A (A2 / A1) and the rate of decrease in the light emission amount of light B (B2 / B1), the first luciferase is Luc2 , Fluc or CBR is preferred, and the second luciferase is preferably CBG or SLR.

The compound represented by the general formula (2) can inhibit the reaction between luciferase and luciferin, and as an inhibitor that inhibits the reaction between the first luciferase and the first luciferin in the light identification method of the present invention. Can be preferably used.
As said 1st luciferin which the compound represented by the said General formula (2) inhibits reaction, it is preferable that it is D-luciferin or its analog.

  In the light identification method of the present invention, the second luminescent substance is luciferin, and the measurement object includes a first luciferin, a first luciferase, the luciferin, and a second luciferase that is reacted with the luciferin, and the luciferin is When the first luciferin is the same substance as the first luciferin, the first luciferin that inhibits the reaction of the compound represented by the general formula (2) is preferably D-luciferin or an analog thereof. At this time, from the viewpoint of increasing the ratio value of the decrease rate of the light emission amount of light A (A2 / A1) and the decrease rate of the light emission amount of light B (B2 / B1), the first luciferase is CBR. The second luciferase is preferably Luc2, Fluc, CBG or SLR.

  Since the compound represented by the general formula (1) or the compound represented by the general formula (2) has a relatively low molecular weight, penetration into cells is likely to occur. Therefore, by using these compounds in the light identification method of the present invention, light generated from the cells of the measurement object can be measured in a non-destructive manner.

  Since the luciferin-luciferase reaction inhibitor of the invention can be suitably used as an inhibitor that inhibits the reaction between the first luciferase and the first luciferin in the light discrimination method of the present invention, it is used as a reagent for dual luciferase assay. be able to. In the color tone control method described later, the luciferin-luciferase reaction inhibitor of the invention can be suitably used as a color tone control reagent.

<Luciferin-luciferase reaction inhibition method>
The luciferin-luciferase reaction inhibitory method of the present invention uses the luciferin-luciferase reaction inhibitor of the present invention.
For example, by adding the luciferin-luciferase reaction inhibitor of the present invention to a reaction system containing the first luciferase and the first luciferin that is the first luminescent substance, the first luciferase and the first luciferin that is the first luminescent substance. Reaction can be inhibited.

The first luciferase and the first luminescent substance can be obtained by adding the luciferin-luciferase reaction inhibitor of the present invention to the reaction system containing the first luciferase, the first luciferin as the first luminescent substance, and the second luminescent substance. It is possible to inhibit the reaction with the first luciferin.
At this time, when the light emission wavelength of the light A generated by the reaction of the first luciferase and the first luciferin is different from the light B generated by the second luminescent substance, the luciferin-luciferase reaction inhibitor of the present invention is used. By adding, the color tone of the light emitted from the reaction system can be changed in the presence of the inhibitor and in the absence of the inhibitor.
The color tone control method using the luciferin-luciferase reaction inhibitor of the present invention includes, for example, a first luciferase, a first luciferin that is a first luminescent substance, and a second luminescent substance, and includes the first luciferase and the first luciferase. An inhibitor that inhibits the reaction between the first luciferase and the first luciferin is brought into contact with a reaction solution in which the light emission wavelengths of the light A generated by the reaction of luciferin and the light B generated by the second luminescent substance are different. The color tone control method can change the color tone of the light emitted from the reaction solution.

<Device>
The apparatus of the present invention is an apparatus for performing the light identification method of the present invention, wherein (a) measuring means for measuring the amount of light emitted from the measurement object, and (b) by the measuring means. Based on the measured value of the luminescence L1 and the value of the luminescence L2 measured by the measuring means, the light A generated by the reaction of the first luciferase and the first luciferin, and the second And a calculating means for identifying the light B generated by the luminescent material. The measuring means may measure a light emission amount or light emission intensity of light emitted from the measurement object.

FIG. 3 is a diagram schematically showing the configuration of the apparatus of the present invention. The apparatus 1 includes a measuring unit 10 and a calculating unit 20. The measuring means 10 measures the light emission amount or light emission amount of light emitted from the measurement object, and is, for example, a photodetector or a photosensor. Note that the measurement object is not a component of the apparatus of the present invention. The measurement means 10 reacts the first luciferase and the first luciferin of the measurement object including the first luciferase, the first luciferin that is the first luminescent substance, and the second luminescent substance, from the measurement object. It is possible to measure the light emission amount L1 of the emitted light. In addition, the measuring means 10 brings an inhibitor that inhibits the reaction between the first luciferase and the first luciferin into contact with the measurement object, and calculates the amount of luminescence generated by the reaction between the first luciferase and the luciferin. It is possible to reduce the light emission amount L2 emitted from the measurement object.
The light emission amount L1 and the light emission amount L2 measured by the measuring unit 10 are calculated by the calculation unit 20 as the light emission amount derived from the light A and the light emission amount derived from the light B among the light emission amounts measured. , Light A and light B can be distinguished. The calculation means is, for example, a computer. With respect to the calculation method of the light emission amount derived from the light A and the light emission amount derived from the light B, those described in the (identification step) in the <light identification method> described above can be given.

  The apparatus of the present invention includes a support unit that supports the measurement object, an inhibitor addition nozzle that adds the inhibitor to the measurement object in order to bring the inhibitor into contact with the measurement object, and holds the inhibitor to the nozzle. And an inhibitor holding unit that supplies the inhibitor, a light emitting amount L1, a light emitting amount L2, a storage unit that stores a decrease rate of the light emitting amount of the first luciferase and the first luciferin, and the like.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

<Inhibition of reactions to various luciferases- (1)>
A reaction solution was prepared by adding 2.5 μL of a luciferase-containing solution (RLL extract), 50 μL of a measurement buffer, and 3 μL of an inhibitor DMSO solution to a reaction vessel and mixing them. The luciferase in the luciferase-containing solution (RLL extract) was synthesized by TnT (registered trademark) Coupled Reticulocyte Lysate Systems (Promega) according to the attached instructions. As the measurement buffer, the LARII buffer attached to the Dual luciferase Assay kit (Promega) was used. The LARII buffer contains D-luciferin. For luciferase, a total of four types of Luc2, Fluc, CBR, and CBG were prepared, and a total of five types of reaction solutions were prepared including a control reaction solution (inhibitor (-)) that did not contain an inhibitor. The inhibitor is a compound represented by the above formula (1-2). The final concentration of the inhibitor in each reaction solution was adjusted to 2 mM. Luminescence generated by the luciferin-luciferase reaction in the reaction solution was measured with a luminometer (AB-2270, manufactured by Atto Corporation).

  The measurement results are shown in FIG. The graph of FIG. 4 shows the relative luminescence activity with the luminescence amount of luminescence obtained from the reaction solution of the inhibitor (−) as 1. The inhibitor did not significantly affect the luminescence activity of CBG, a green luminescence luciferase, and the luminescence activity of CBG was about 0.8, but the inhibitor decreased the luminescence activity of Luc2 and Fluc to about 0.3. The luminescence activity of CBR was reduced to about 0.1.

<Inhibition of reactions to various luciferases- (2)>
A reaction solution was prepared by adding 2.5 μL of a luciferase-containing solution (RLL extract), 50 μL of a measurement buffer, and 3 μL of an inhibitor DMSO solution to a reaction vessel and mixing them. The luciferase in the luciferase-containing solution (RLL extract) was synthesized by TnT (registered trademark) Coupled Reticulocyte Lysate Systems (Promega) according to the attached instructions. As the measurement buffer, the LARII buffer attached to the Dual luciferase Assay kit (Promega) was used. The LARII buffer contains D-luciferin. For luciferase, a total of four types of Luc2, Fluc, CBR, and CBG were prepared, and a total of five types of reaction solutions were prepared including a control reaction solution (inhibitor (-)) that did not contain an inhibitor. The inhibitor is a compound represented by the above formula (2-2). The final concentration of the inhibitor in each reaction solution was adjusted to 100 μM. Luminescence generated by the luciferin-luciferase reaction in the reaction solution was measured with a luminometer (AB-2270, manufactured by Atto Corporation).

  The measurement results are shown in FIG. The graph of FIG. 5 shows the relative luminescence activity with the luminescence amount of luminescence obtained from the reaction solution of the inhibitor (−) as 1. The inhibitor hardly affected the luminescence activity of CBG, which is a green luminescence type luciferase, and the luminescence activity of CBG remained at about 1. The inhibitor decreased the luminescence activity of Luc2 and Fluc to about 0.8 and the luminescence activity of CBR to about 0.1.

<Transient gene transfer into spinach>
Regarding the light-inducible promoter CAB1, which is known to be induced by light, the promoter activity under different light conditions was confirmed.
First, a light-inducible promoter CAB1 and CBG fusion gene (CAB-CBG), a cauliflower mosaic virus 35S promoter CaMV-35S and CBR fusion gene (35S-CBR), and a CaMV-35S and CBG fusion gene (35S). -CBG) was produced by the method described in Ogura et al., Plant Biotechnology 22 (2), 151-155 (2005) (Non-patent Document 2).
The CAB1 promoter is a promoter of a target gene whose gene expression level is to be measured, and the 35S promoter is a promoter serving as an internal control.

Subsequently, these fusion genes were simultaneously introduced into spinach cells using a gene gun. The introduction conditions are based on Ogura et al., Plant Biotechnology 22 (2), 151-155 (2005) (Non-patent Document 2). The cells into which the fusion gene has been introduced are divided into a light condition treatment group and a dark condition treatment group. The light condition treatment group is treated under continuous white illumination for 12 hours (50 μmol− ² sec ⁻¹ ), and the dark condition treatment group. Were treated with a continuous dark period of 12 hours. A cell extract was prepared using the PLB buffer attached to Dual luciferase Assay kit (manufactured by Promega).
Cell extract is
-CAB-CBG and 35S-CBR-introduced cells-Under light conditions-CAB-CBG and 35S-CBR-introduced cells-Under dark conditions-35S-CBG and 35S-CBR-introduced cells-Under light conditions-35S-CBG and 35S-CBR-introduced cells under dark conditions,
A total of 4 types were obtained.
10 μL of each of the above cell extracts and a measurement buffer were added to the reaction vessel to obtain a reaction solution. As the measurement buffer, the LARII buffer attached to the Dual luciferase Assay kit (Promega) was used. The LARII buffer contains D-luciferin. The light emission amount (light emission amount L1) emitted from each reaction solution was measured with a luminometer (AB-2270, manufactured by Atto Corporation). Next, an inhibitor was added to each reaction solution, and the luminescence amount (luminescence amount L2) emitted from each reaction solution was measured using a luminometer. The inhibitor is a compound represented by the above formula (1-2).

  The rate of decrease in luminescence due to the reaction between CBR and D-luciferin is 10%, and the rate of decrease in luminescence due to the reaction between CBG and D-luciferin is 100% (no inhibition). ), The amount of luminescence due to the reaction between CBR and D-luciferin in the absence of an inhibitor in each reaction solution and the amount of luminescence due to the reaction between CBG and D-luciferin were determined.

The results are shown in FIG. The graph of FIG. 6 divides the value of the amount of luminescence produced by the reaction between CBG and D-luciferin in the absence of an inhibitor in each reaction solution by the value of the amount of luminescence produced by the reaction between CBR and D-luciferin. Value.
In the graph of FIG.
-CAB-CBG and 35S-CBR-introduced cells-The above values for the reaction solution containing cell extracts treated under bright conditions are "CAB1-Light",
-CAB-CBG and 35S-CBR-introduced cells-The above values for the reaction solution containing cell extract treated under dark conditions are "CAB1-Dark",
-35S-CBG and 35S-CBR-introduced cells-The above value for a reaction solution containing a cell extract treated under bright conditions is "35S-Light"
-35S-CBG and 35S-CBR-introduced cells-The above value for a reaction solution containing a cell extract treated under dark conditions was expressed as "35S-Dark".

In this way, the value of the promoter activity normalized by the internal standard could be calculated.
From the graph of FIG. 6, it can be seen that the activity of the light-inducible promoter CAB1 is increased under light conditions than under dark conditions.
From the above, even when the light from the reaction of CBR and D-luciferin and the light from the reaction of CBG and D-luciferin are detected without distinction from the cell extract of the measurement object, the amount of luminescence Both lights can be distinguished based on the value of L1 and the value of luminescence L2, and it was demonstrated that a reporter assay using two types of luciferases can be performed.

  The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention.

The present invention can be applied to any activity evaluation using luciferase luminescence. In addition to gene expression quantification techniques such as the dual luciferase method using a luciferase reporter, there are applications to enzyme activity quantification techniques or microbial detection techniques, and application to drug discovery techniques using them is also conceivable.
It can also be used as a general technique for changing the color tone of luciferase luminescence without changing the composition of multicolor luciferase.

DESCRIPTION OF SYMBOLS 1 ... Apparatus, 10 ... Measuring means, 20 ... Calculation means

Claims (3)

A luciferin-luciferase reaction inhibitor, which is a compound represented by the following general formula (1), a compound represented by the following general formula (2), or a salt thereof.

[In Formula (1),
X ¹ represents CH or N, R ¹ represents a halogen atom, and R ² represents a linear or branched alkyl group having 1 to 4 carbon atoms.
n represents the number of R ¹ and is an integer of 0 to 3. When n is 2 or more, R ¹ may be the same or different from each other. m represents the number of R ² and is an integer of 0 to 2, and when m is 2, R ² may be the same as or different from each other. ]

[In Formula (2),
X ² represents CH or N, R ³ represents a halogen atom, R ⁴ represents a single bond or a divalent linking group, and R ⁵ represents a linear or branched alkyl group having 1 to 4 carbon atoms. Represents.
p represents the number of R ³ and is an integer of 0 to 3, and when p is 2 or more, R ³ may be the same or different from each other. q represents the number of R ⁵ , and is an integer of 0 to 2, and when q is 2, R ⁵ may be the same as or different from each other. ]   A method for inhibiting a luciferin-luciferase reaction, comprising using the inhibitor according to claim 1 which inhibits a reaction between luciferase and luciferin. The following steps:
The first luciferase, the first luciferin, which is a measurement target of the luminescence level and reacts with the first luciferase to emit light A when measuring the luminescence level, and the first luciferin, which is the measurement target of the luminescence level, and light when measuring the luminescence level. A measurement object including a second luminescent substance that emits B (excluding a fluorescent substance) (provided that the second luminescent substance is luciferin, and the measurement object is a first luciferin, a first luciferase, the luciferin, And the second luciferase to be reacted with the luciferin is different from the first luciferase and the second luciferase), the first luciferase and the first luciferin are reacted, and emitted from the measurement object. A first measurement step of measuring the emitted light amount L1
An inhibitor that inhibits the reaction between the first luciferase and the first luciferin is brought into contact with the measurement object, and the amount of luminescence generated by the reaction between the first luciferase and the luciferin is reduced. A second measuring step for measuring the emitted light amount L2,
The value of the light emission amount L1, the value of the light emission amount L2, the rate of decrease in the light emission amount of the first luciferin that is the first light emitting substance by the inhibitor acquired in advance, and the inhibitor acquired in advance. The light emission amount of light A generated by the reaction of the first luciferase and the first luciferin based on the decrease rate of the light emission amount of the second light emitting material, and the light emission of light B generated by the second light emitting material A discriminating step for calculating the amount and identifying the light emission amount of the light A and the light emission amount of the light B;
A kit for performing a light identification method comprising the inhibitor according to claim 1.

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