JP2003035714A - Method for detection of existence of binding capacity of specimen to protein and marker protein generated in solution using expression vector used in the same as well as method of preparing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method which detects the existence of the binding capacity of a specimen to a protein and to provide a method in which the action of the specimen especially on a hormone receptor can be analyzed precisely. SOLUTION: The method which detects the existence of the binding capacity of a specimen to a protein is provided with a process (1) in which the protein labeled with a fluorescent material is made to exist in a solution and a process (2) in which the specimen is made to act on the labeled protein described in the process (1) while the fluorescent intensity from the fluorescent material is being measured continuously and in which the existence of the binding capacity of the specimen to the protein is determined on the basis of a change in the obtained continuous fluorescent intensity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting the presence or absence of the binding ability of a test substance to a protein, especially a hormone receptor, and a method for producing the same in a solution using an expression vector. The present invention relates to a labeled protein and a method for producing the labeled protein.

[0002]

2. Description of the Related Art Conventionally, research on protein functional analysis has been mainly conducted on the analysis of gene sequences encoding proteins. However, due to the rapid progress of human genome analysis in recent years, the direction of protein functional analysis research is shifting from the analysis of genes existing in cells to the dynamics of proteins synthesized from such genes and the analysis of their functions. I am doing it.

In vivo, various molecules having specific functions (for example, functional proteins such as enzymes and receptors; proteins that retain cell structure; and biological proteins such as lipids, sugar chain proteins, and ions) (Active molecule, etc.) constantly fluctuates in response to various gene expressions and signal transduction, and such fluctuations maintain the life mechanism. For example, in order to accurately observe and analyze the biological function of a protein, it is necessary to observe the dynamics of the protein that maintains a three-dimensional structure capable of exhibiting the originally desired function at the molecular level. As a means for that purpose, conventionally, a method of labeling a target protein with a labeling substance and tracking it is generally used.

For example, one of the conventional methods for labeling a target protein is a fluorescent labeling method (Cytometry 2000 Dec 1; 41.
(4): 316-20: Lipopolysaccharide (LPS) labeled with Al
exa 488 hydrazide as a novel probe for LPS binding
studies. Triantafilou K, Triantafilou M, Fernandez
N.). This is a method of nonspecifically adsorbing a fluorescent substance such as FITC (that is, fluorescein isothiocyanate) and Alexa onto a protein. However, such a fluorescent labeling method can
When analyzing the interaction between a protein and another substance such as an antibody reaction, it is difficult to obtain an accurate analysis result because the three-dimensional structure of the protein in question is likely to be displaced.

Further, as a conventional method superior in specificity and sensitivity to the above-mentioned fluorescence labeling method, there is a method of producing a target protein using a transgenic cell. For example, a method using fluorescent green protein (hereinafter abbreviated as GFP) as a labeling substance is described in (Biotechniques 1995 Oct; 19 (4): 650-5 R
elated Articles, Books Green fluorescent proteinas
a reporter of gene expression and protein localiz
ation. Kain SR, Adams M, Kondepudi A, Yang TT, War
d WW, Kitts P.). In this method, a gene encoding a target protein and a gene encoding GFP are incorporated into the same vector, and this is introduced into a cell, and a fusion protein of GFP and the target protein is expressed to obtain a labeled target protein. is there. Here, it is already known that GFP used as a labeling substance is a biomolecule derived from Owan jellyfish and has no biotoxicity. Further, the binding of GFP to the target protein can be selectively carried out at the ends of the protein. Therefore, by using such a technique, the behavior of the target protein can be observed while the physiological structure of the target protein is retained.

The means for observing the intracellular behavior of the target protein fluorescently labeled by such a method is generally observed by a phase contrast microscope or a differential interference microscope. However, the resolution of these microscopes is at most 0. It is about several μm. Therefore, even though it is sufficient for observing the microscopic structure in living cells as an image, it is not sufficient for finely analyzing protein interactions.

[0007] Alternatively, the behavior of such a protein of interest in the cell is the fluorescence correlation spectroscopy (hereinafter abbreviated as FCS; Fluorescence Correlation Spe) developed in recent years.
You can also use ctroscopy). FCS
Is a method of calculating a physical quantity such as the number and size of molecules by measuring the fluctuation motion of a fluorescently labeled target molecule in a medium and applying the measured fluctuation to an autocorrelation function. By using such a method, by determining the physical quantity such as the number and size of the molecules of the fluorescently labeled protein present in the measurement system, the existing state thereof is continuously measured,
Thereby, the interaction between the target protein and other molecules can be analyzed finely.

Furthermore, as a biological application example of the above-mentioned FCS, FC in which GFP introduced into living cells is measured
The method of S measurement has been reported (Proc.Natl.Acad.Sci.U
SA vol96, pp10123-10128, 1999). According to the literature,
It has been pointed out that even in the steady state, where no signal is received, association with molecules of multiple sizes within the cell occurs. That is, it is very difficult for the conventional FCS method to stably measure the target molecule in the state existing in the cell and achieve accurate analysis.

[0009]

In view of the above circumstances, an object of the present invention is to accurately analyze a method for detecting the presence or absence of the binding ability of a test substance to a protein, particularly the action of the test substance on a hormone receptor. It is to provide a possible method.

In particular, it is an object of the present invention to provide a method and an apparatus capable of accurately detecting the action of a test substance on a hormone receptor by stably analyzing the behavior of the fluorescent labeled molecule by utilizing the fluorescent molecule measurement by FCS. Specifically, it is possible to stably fluoresce a fluorescent substance labeled on a biomolecule to be analyzed, reduce noise by reducing meaningless association with intracellular molecules, and to add biomolecules in a solution. The object is to provide a detection system that is constructed stably and is simple and highly reproducible.

Another object of the present invention is to provide a labeled protein produced in solution using an expression vector for use in the above method and a method for producing the labeled protein.

[0012]

[Means for Solving the Problems] In order to solve the above-mentioned problems, first of all, in order to achieve a detection system capable of eliminating association with meaningless molecules in cells, It was thought that it was necessary to extract the target protein from the above and to make it exist in the solution in a uniform state. Based on this idea, as a result of earnest research, in vitro translati
on / expression; cell-free transcription method) was used to make it possible to produce a fusion protein of a fluorescent protein and a desired protein (eg, hormone receptor) in a test. Using this, a single means was created in which there is no impurity causing intracellular association, and a means for solving the above problems was found.

That is, the present invention is as follows: A method for detecting the presence or absence of the binding ability of a test substance to a protein; (1) the presence of a protein labeled with a fluorescent substance in a solution; (2) the fluorescence intensity from the fluorescent substance is continuously applied. Of the labeled substance according to (1) while performing the quantitative measurement, the test substance is allowed to act on the labeled protein, and the binding ability of the test substance to the protein is determined based on the resulting change in fluorescence intensity over time. Determining the presence or absence of 1 .; A method for detecting the presence or absence of the ability of a test substance to bind to the protein according to 1 above, further comprising (3) (2) the change in the fluorescence intensity over time and the test substance 2. determining the presence or absence of the binding ability of the test substance to the protein based on the comparison with the change in the fluorescence intensity over time obtained in the absence thereof; The detection method according to any one of 1 or 2 above, wherein (1) includes (a) a gene encoding the protein, and a gene encoding a fluorescent substance for labeling the protein; Constructing an expression vector by incorporating a promoter for expressing a gene encoding the protein extracellularly into a vector; and (b) under the conditions that allow expression and transcription of the gene and protein production described in (a). 3. The step of causing the expression vector to exist in a solution to produce a protein labeled with the fluorescent substance; 1 above, wherein the protein is selected from the group consisting of hormone receptors, antigens and antibodies
4. The method according to any one of 1 to 3; 5. The detection method according to any one of 1 to 3 above, wherein the protein is an estrogen receptor; 4. The detection method according to the above 3, wherein the protein is estrogen receptor β, the fluorescent substance for labeling is fluorescent green protein, and the promoters are T3 and T7.
Or a detection method characterized by being an expression vector selected from the group consisting of SP6; 7. The detection method according to any one of 1 to 6 above, wherein the measurement and the determination are performed by fluorescence correlation spectroscopy; A method for producing a fluorescently labeled protein, comprising: (a) expressing a gene encoding the protein, a gene encoding a fluorescent substance for labeling the protein, and a gene encoding the protein in a solution. Constructing an expression vector by incorporating a promoter for the expression into a vector; (b) allowing the expression vector to exist in a solution under conditions that allow expression and transcription of the gene described in (a) and protein production, and 8. Producing a protein labeled with a fluorescent substance; 9. The method for producing a labeled protein according to 8 above, wherein the protein is estrogen receptor β,
10. The production method, wherein the labeling fluorescent substance is a fluorescent green protein, and the promoter is an expression vector selected from the group consisting of T3, T7 or SP6; A labeled protein produced by the production method described in 8 or 9 above.

[0014]

DETAILED DESCRIPTION OF THE INVENTION Method for detecting presence / absence of binding ability of test substance to protein 1. Outline According to the present invention, a method for detecting the presence or absence of the binding ability of a test substance to a protein is provided.

The term "protein" as used herein refers to proteins such as hormone receptors, antigens and antibodies, and has a property of specifically binding to a specific substance. That is, it is preferable to use the present invention to detect a substance that specifically binds to such a protein. Hereinafter, an embodiment using a hormone receptor will be described, but the present invention is not limited to the hormone receptor, and can be similarly applied to other proteins.

As a preferred requirement in the present invention, a protein means a protein that should exist in cells in nature. Therefore, in the present invention, a reaction with a test substance in a solution is carried out by taking out a desired protein from cells or by allowing an artificially produced protein to exist in an appropriate solution. . Reacting a protein that could not have been in solution in solution favors the binding reaction and the conditions necessary for the assay and allows application to the desired assay. When there is a demand to continuously monitor and measure the binding reaction between a protein and a test substance, it is convenient to measure the change due to the binding reaction by a morphological or kinematic method. Morphological techniques are also possible when both the protein and the test substance have morphological characteristics, but many involve very small morphological changes, so it is important to measure kinematic changes. It is preferable for improving the measurement accuracy.

2. Method for detecting presence or absence of action of test substance on hormone receptor In one aspect of the present invention, a method for detecting presence or absence of action of test substance on a hormone receptor is provided.

“Analyte” as detected in accordance with an aspect of the present invention means a chemical substance other than the original ligand that acts on the desired hormone receptor. Therefore, for any chemical suspected of endocrine disrupting properties,
It is possible to test according to the invention.

As used herein, the term "action of the test substance on the hormone receptor" refers to the action of the test substance on the desired hormone receptor and mimics the physiological action caused by the original ligand of the hormone receptor. Exhibits an action, for example, that the test substance binds to the ligand binding region of the desired hormone receptor, that is, that the test substance shows an affinity for the ligand binding region of the desired hormone receptor, and As a result of such binding, the test substance affects the existence state of the hormone receptor. Here, “the test substance affects the existing state of the hormone receptor” means that the size of one molecule of the receptor is changed by the binding of the test substance to the receptor. For example, the test substance is usually 1
It also includes the action of binding to a hormone receptor present in a dimer, thereby causing the hormone receptor to dimerize. As used herein, the term "in vitro" is used for convenience and specifically refers to an in vitro assay system that does not necessarily require cells or does not contain cells (ie, cell-free). Therefore, it does not literally indicate only "the inside of the test tube". The detection method and the production method according to the present invention can be carried out in any of test tubes, beakers, microtubes, multiwell plates and other containers, and a plane capable of holding a required amount of liquid. In addition, in the present specification, the term "in solution" is also used synonymously with the term "in vitro" used herein.

According to an embodiment of the present invention, the detection method comprises
While continuously measuring the fluorescence intensity from the protein labeled with the fluorescent substance, the presence or absence of the change and / or the amount obtained when the test substance is allowed to act is used to determine the binding ability of the test substance. Any determinable measurement method, preferably fluorescence correlation spectroscopy (hereinafter referred to as FCS), is used. That is, a hormone receptor labeled with a fluorescent substance is allowed to exist in a solution, a test substance is allowed to act on the labeled hormone receptor, and the state of molecules present therein is controlled by FC
Analyze by S.

3. Fluorescent Labeled Hormone Receptor The fluorescent labeled hormone receptor used according to the present invention is a hormone receptor labeled with a fluorescent substance.

The hormone receptor used according to the present invention may be either a receptor present in the cell membrane, a receptor present in the cytoplasm or a receptor present in the nucleus. In particular, the receptor present in the cytoplasm is preferable, and the receptor present in the nucleus is more preferable.

Examples of preferred hormone receptors include proteins belonging to the nuclear hormone receptor superfamily such as estrogen receptor, progesterone receptor, thyroid hormone receptor and glucocorticoid receptor. Particularly preferred receptors are estrogen receptors, which are human estrogen receptor α (hereinafter also referred to as hERα) and human estrogen receptor β (hereinafter also referred to as hERβ).

As will be described later, the FCS technique is a technique for performing a desired analysis based on changes in the size of molecules. Therefore, it is desirable that the molecular size of the hormone receptor changes before and after the action of the original ligand or the test substance on the hormone receptor. For example, the size of the molecule as a whole increases due to the binding of a ligand or a test substance to the hormone receptor, or the size of the molecule changes, or the hormone receptor exists as a monomer before the action of the ligand. Receptors that dimerize or multimerize by the action of a ligand or a test substance are particularly preferable.

For example, the estrogen receptor forms a dimer when a ligand binds to the estrogen receptor. The activity of estrogen in cells under physiological conditions is caused as follows; binding of estrogen to an estrogen receptor to form a complex, dimerization of the complex, and the dimerization The body binds to an estrogen response element located upstream of the target gene, and further the coactivator binds to the dimer, whereby the gene product is transcribed and translated. Thus, hormone receptors whose receptors are dimerized during the course of the estrogen action are particularly preferred for use according to the invention.

The fluorescent substance for labeling the hormone receptor protein used according to the embodiment of the present invention exists as a protein, and can emit a light signal including fluorescence and light emission by laser irradiation or its own energy. desirable. In nature, even GFP extracted from Owan jellyfish is a modified form of YF.
It may be P, CFP or RFP. Further, a similar fluorescent protein derived from Renilla may be used. Such fluorescent proteins include, for example, green fluorescent protein (hereinafter referred to as GFP).
Green Fluorescent Protein), CFP (Cyan
Fluorescent Protein), YFP (Yellow Fluorescent
Protein) and RFP (Red Fluorescent Protein)
Etc. These fluorescent proteins can be expressed as a fusion protein in the form of being fused with the hormone receptor protein by using a DNA fragment having a gene sequence that encodes and using a well-known gene recombination technique as described later.

4. Method for Producing Fluorescently Labeled Hormone Receptor The fluorescently labeled hormone receptor used in accordance with the present invention is produced in vitro using an expression vector constructed using a gene recombination technique.

The conventional GFP vector has a mechanism in which a promoter sequence for activating transcription of a desired protein functions only in cells. Conventionally, when such a vector is used, it is necessary to introduce the gene into a mammalian cell or microorganism and use the RNA transcription activating enzyme of the host. The inventors of the present invention have added RNA required for RNA transcriptase synthesis upstream of a gene encoding a desired protein in such a fluorescent protein vector.
A promoter was introduced. By allowing such an expression vector to be present in an appropriate cell-free system together with the corresponding enzyme, the gene in the expression vector is transcriptionally activated, RNA synthesis becomes possible, and a desired protein is produced.

The vector used according to the invention is RN
A vector having an A polymerase promoter sequence. Further, the promoter used according to the present invention is
For example, promoters for RNA polymerases such as T3, T7 or SP6.

In order to synthesize a desired fluorescent labeled hormone receptor in vitro, first, an expression vector into which a desired gene is introduced is constructed. For example, in a vector containing a gene having a promoter sequence for expressing a desired gene under cell-free conditions and a restriction enzyme site that can be used for gene transfer, encode a gene encoding a fluorescent protein and a desired hormone receptor. All you have to do is incorporate the gene.

Specifically, for example, an expression vector can be constructed by preparing a fusion gene of a gene encoding a fluorescent protein and a gene encoding a desired hormone receptor and incorporating the gene into a desired vector. For example, pSPOR having a T7 RNA promoter sequence
T1 (Lifetech Oriental, plasmid pSP
A gene encoding a desired hormone receptor protein may be fused to ORT1) downstream of the GFP gene and incorporated into the upstream of the GFP gene.

For example, in the fusion of the above-mentioned hormone receptor-encoding gene with a fluorescent protein-encoding gene, the desired hormone receptor gene is first cloned, and the fluorescent protein gene and the receptor gene previously incorporated into an expression vector are combined. Can be ligated in an expression vector.

For ligation, the fluorescent protein gene and the receptor gene are effectively used with arbitrary multi-cloning site sequences. This sequence is not particularly limited as long as it is a sequence that allows cleavage with a restriction enzyme and ligation with a ligase, and has a length that does not hinder the expression of the fusion protein. Here, in order to ligate the fluorescent protein gene previously incorporated in the expression vector and the receptor gene, the multi-cloning site held by the expression vector plays a role of a seam.

It is convenient to use a commercially available expression vector in which the desired promoter or fluorescent protein gene has been previously incorporated. The gene encoding the hormone receptor protein used in accordance with the present invention may include at least the coding region encoding the amino acid sequence of the protein, that is, the gene sequence from the start codon to the stop codon, and is useful for expression of the hormone receptor protein. An upstream sequence of the start codon and a downstream sequence of the stop codon can be included in any length as long as they do not interfere.

For example, in the case of the human estrogen receptor β gene (the cDNA sequence is shown in SEQ ID NO: 1), it suffices if it contains at least the nucleotide sequence at positions 99 to 1688 which is the coding region. The fusion gene actually produced in the present invention contains the sequence of positions 53 to 1735 of the hERβ gene, as described in Examples below. However, the length of the hERβ gene sequence within the fusion gene varies depending on the cloning conditions of the hERβ gene and the conditions for producing the fusion gene.
The sequence length of the Rβ gene should not be limited to that produced in the Examples below.

According to the present invention, the gene encoding the fluorescent protein fused with the above-mentioned hormone receptor gene is
There is no particular limitation as long as it is a gene of a substance that emits detectable fluorescence, and a substance that emits fluorescence, for example, GFP, YF
A gene encoding any of P, RFP and the like may be used. The gene encoding the fluorescent protein may be of any length including the coding region of the gene, as long as the gene can express the fluorescent protein.

The production of the fluorescently labeled hormone receptor protein in vitro by the expression vector constructed as described above can be carried out by the in vitro expression method using a generally used in vitro expression system. The genes in the expression vector may be arranged in the order of promoter, receptor, fluorescent protein, or promoter, fluorescent protein, receptor.

The hormone receptor produced in this manner can exist while maintaining the morphology observed in the state in which it is originally present in the cell. Further, such a fluorescent labeled hormone receptor can be stored as an expression vector. Therefore, if synthesized from the expression vector at the start of the test, denaturation during storage can be avoided. Also, when generated in a test tube like this,
It is possible to remove associated molecules that are factors of association during FCS measurement, such as cell skeletal proteins and lipids that constitute various organs. The details regarding the FCS will be described later.

The thus-produced fluorescently labeled hormone receptor is also included in the scope of the present invention. The produced fluorescent-labeled hormone receptor may have one or several amino acids deleted, substituted or added in its amino acid sequence as long as it retains the original physiological activity and three-dimensional structure. Further, a method for producing a fluorescently labeled hormone receptor in a test using such an expression vector is also included in the scope of the present invention. Furthermore, the expression vector is also included in the scope of the present invention.

The fluorescently labeled hormone receptor produced in this way may be used without further purification, or may be purified and used or stored as follows. For example, such purification can be performed by any of the per se known purification means used for proteins.

5. Fluorescence Correlation Spectroscopy Fluorescence Correlation Spectroscopy (ie FC
S) is a measurement of the fluctuation motion of a fluorescently labeled target molecule in a medium, and the autocorrelation function
is a technique for accurately measuring the micromotion of individual target molecules by using (ion) (Reference: D. Magde and E.
Elson, “Fluorescence correlation spectroscopy. I
I. An experimental realization ”, Biopolymers 1974
13 (1) 29-61).

The FCS used in accordance with the present invention analyzes the Brownian motion of fluorescent molecules in a solution in a microscopic area by a laser confocal microscope to analyze the diffusion time from the fluctuation of the fluorescence intensity to determine the physical quantity (number of molecules, It is performed by measuring the (size). Analysis by FCS that captures molecular fluctuations in such a minute region is effective for highly sensitive and specific detection of intermolecular interactions.

The principle of detection by FCS implemented according to the present invention will be described in more detail. In FCS, a fluorescence signal generated from a microscopic field area in a sample is detected and quantified by a microscope. At this time, the fluorescently labeled target molecule in the medium is constantly moving (that is, Brownian motion). Therefore, the detected fluorescence intensity changes depending on the frequency with which the target molecule enters this microscopic visual field region and the time for which the target molecule remains in the microscopic visual field region. For example, if dimerization occurs and the apparent molecular weight increases, the movement of the target molecule slows down and the apparent number of molecules decreases. As a result, the frequency of entering the microscopic visual field region is reduced, and the observed fluorescence intensity is changed. By monitoring such changes in fluorescence intensity, changes in the apparent molecular weight of the target molecule can be tracked.

6. Fluorescence Correlation Analyzer An example of a fluorescence correlation analyzer that can be used in the method of the present invention will be described below with reference to FIG. As shown in FIG. 1, the fluorescence correlation spectroscopy apparatus includes a laser light source 1, a light intensity adjusting means (an ND filter here) 2 for reducing the intensity of a light beam from the laser light source 1, and an appropriate light intensity adjustment. A light attenuation selection device (here, an ND filter changer) 3 for setting the means 2, and optical systems 4 and 5 for converging the light beam from the laser light source 1 on the sample to form a confocal region, and fluorescent molecules 6 with a sample containing
And optical systems 7 to 11 for collecting fluorescence from the sample,
It comprises a photodetector 12 for detecting the collected fluorescence and a fluorescence intensity recording means for recording the change in the fluorescence intensity. Thus, the fluorescence correlation analyzer uses a confocal laser microscope. In FIG. 1, the laser light source (1)
May be an argon laser, a helium-neon laser, krypton, helium-cadmium, or the like.

In FIG. 1, the optical systems 4 and 5 for converging the light beam from the laser light source on the sample to form the confocal region specifically mean the dichroic mirror 4 and the objective lens 5. The light beam from the laser light source 1 is first attenuated according to the attenuation degree of the fluorescence intensity adjusting means (here, the ND filter) 2 along the path shown by the arrow in FIG. 1, and then the dichroic mirror 4 is used.
Refracts in the direction of the stage at 90 degrees to the incident light,
The sample on the stage 6 is irradiated through the objective lens 5. In this way, the light beam is focused on the sample at one minute point to form a confocal region.

In the present invention, the stage 6 sample may be a solution in which fluorescent molecules are suspended, or a biological component such as a protein labeled with fluorescent molecules. In addition to chemical substances, fluorescent molecules include green fluorescent proteins (that is, GF
It can be prepared by a method of expressing a fusion protein of a fluorescent protein and a protein to be analyzed, using a known genetic engineering method such as P).

In FIG. 1, the optical systems 7 to 11 for collecting the fluorescence emitted from the fluorescent molecules in the confocal region are, specifically, the filter 7, the tube lens 8 and the reflecting mirror 9.
After refracting by the lens and storing in the pinhole 10, the lens 1
The light passes through 1 and is focused on the photodetector 12.

A photodetector (here, avalanche photodiode) 12 for detecting the collected fluorescence converts the received light signal into an electric signal and sends it to the fluorescence intensity recording means (here, computer) 13. To do.

The fluorescence intensity recording means 13 for recording the change in fluorescence intensity records and analyzes the transmitted fluorescence intensity data. Specifically, the autocorrelation function is set by analyzing the fluorescence intensity data. An increase in the molecular weight and a decrease in the number of molecules due to the movement of the fluorescent molecule (for example, dimerization of the fluorescent molecule), or a decrease in the number of molecules due to the binding of the fluorescent molecule to a specific DNA region are detected by a change in the autocorrelation function. can do.

An apparatus for performing the above-mentioned FCS is also included in the scope of the present invention.

7. Method for detecting the presence or absence of the action of the test substance on the hormone receptor The fluorescence labeled hormone receptor synthesized as described above and the test substance are reacted under appropriate conditions, and the molecular intensity is measured by FCS using the fluorescence intensity as an index. Then, the presence or absence of the action of the test substance on the hormone receptor can be detected by calculating the change in the autocorrelation function based on it. The presence or absence of the action of the test substance on the hormone receptor may be determined by comparing the data before the reaction and the data after the reaction, or the data obtained in the presence of the test substance and the test substance The presence or absence of the action of the test substance on the hormone receptor may be determined by comparing the data obtained in the absence thereof.

Predetermined conditions and appropriate conditions for detecting the action of the test substance on the fluorescent-labeled hormone receptor, such as reaction temperature, reaction time, and composition of reaction solution, are determined according to the fluorescent-labeled hormone receptor used and the target. The practitioner can arbitrarily select according to the test substance.

According to the present invention, when a test substance is added to a desired fluorescence-labeled hormone receptor and maintained under appropriate reaction conditions set in advance, the test substance has an action on the hormone receptor. If so, the test substance binds to the hormone receptor. As a result, a series of biochemical reactions similar to the case of the original ligand occurs, the state of the receptor is changed, and thereby,
The fluorescence intensity detected by FCS changes. Whether or not the test substance has an action on the hormone receptor is determined using such changes as an index. If there is no change in the fluorescence intensity detected before and after the addition of the test substance, it is determined that the test substance has no effect on the hormone receptor.

In the above, the method for detecting the presence or absence of the action of the test substance on the hormone receptor has been described as the embodiment of the present invention. However, the present invention is not limited to hormone receptors. That is, similarly,
A protein such as an antibody or an antigen is produced by an expression vector (at this time, it can be produced as a fusion protein with a fluorescent labeling substance), and a substance which specifically binds to such protein is
It is also possible to detect by CS technology.

[0055]

[Example] 1. Preparation of Recombinant Protein for Confirming Function of GFP + Estrogen Receptor β Fusion Protein Experimental Purpose In this experiment, it was confirmed by in vitro expression method that the desired protein was expressed from the cloned GFP + estrogen receptor (ER) β gene. And GFP
The purpose of the present invention is to confirm in vitro whether the + estrogen receptor β protein forms a dimer and to confirm the function of the GFP + estrogen receptor β protein.

Experimental Method Cloned GFP + Estrogen Receptor β
Transcription / translation system for vectors for in vitro expression containing the gene for Escherichia coli and the gene for estrogen receptor β only
slation system, TNT Quick Coupled Trans from Promega
Cription / Translation System) was used to conduct an expression experiment of the GFP + estrogen receptor β gene and the gene product of the estrogen receptor β gene,
The antigenicity is confirmed by Western blotting and the like, and the dimer formation function is confirmed.

Experimental Procedures and Results 1) Construction of In Vitro Expression Vector As a vector for expressing a protein using the in vitro transcription / translation system, pSPORT1 (Lifetech Oriental Co., Ltd.,
The plasmid pSPORT1) was chosen. This vector has a T7 RNA promoter sequence necessary for RNA synthesis and a restriction enzyme site restriction site (Sall, BamHI) downstream of which the GFP + ERβ fusion gene can be introduced. At these sites, it was decided to incorporate the GFP + ERβ fusion gene (Fig. 2a) and the ERβ gene (Fig. 2b), which were excised from the vector constructed for cell expression and purified by electrophoresis (Fig. 2).

It was prepared using pEGFP + ERβ containing the GFP gene + ERβ fusion gene, isolated, and cut with a restriction enzyme.

The cleaved plasmid was confirmed by electrophoresis, and after a large amount of electrophoresis, GF was prepared by gel excision method.
The P + ERβ fusion gene and the cleavage fragment of the ERβ gene were cut out and the DNA extraction method using glass beads (Bio101,
The cleaved fragment was purified from the gel with Geneclean III). After purification, the concentration and the degree of purification were confirmed by electrophoresis. Similarly, the plasmid pSPORT1 vector having the T7 promoter sequence was also cleaved with a restriction enzyme and purified by a DNA extraction method using glass beads. The concentration was confirmed by electrophoresis (Fig. 3).

Cleaved pSPORT1 vector and GFP
+ ERβ fusion gene and truncated pSPORT1
The vector and the ERβ gene were mixed together, ligated, transformed into E. coli DH5α, and then cultured on Amp + plate.

After selecting colonies and picking them up in a PCR tube, PCR was carried out using a primer for ERβ, and amplification was confirmed by electrophoresis.

PSPORT1 vector + GFP + ERβ
Recombinants of 4 clones or more with the fusion gene and 3 clones or more of the pSPORT1 vector + ERβ gene could be obtained (FIG. 4). Plasmids were isolated from these clones and cleaved with several restriction enzymes, and it was confirmed that the obtained plasmids were correct recombinants (Figs. 5 and 6). By the above operation, a plasmid containing the GFP + ERβ fusion gene (FIG. 5) and the ERβ gene (FIG. 6) that can be used for in vitro expression could be obtained.

2) In vitro expression The protein was expressed in an in vitro expression system using the constructed expression vector. For expression, Promega's TNT Quick Coupled Transcription / Translation System
nslation System) was used. In addition, tR having a biotin-labeled amino acid lysine at the time of protein synthesis during expression
Biotin-labeled lysine was incorporated into the expressed and synthesized protein by adding NA (Tr of Promega).
anscend Non-Radioactive Translation Detection Syst
use em). As a control for the in vitro expression system, the luciferase gene attached to the kit was expressed.
The reaction solution after in vitro expression was separated by SDS polyacrylamide gel, stained with bromophenol blue, or transferred with Western blot, and then streptavidin-
Detected by color reaction on an alkaline phosphatase-based membrane (Proc's Transcend Non-Radioactive Transl
ation Detection System).

Results: When the reaction solution for in vitro expression was separated by SDS polyacrylamide gel and stained with bromophenol blue, the bands of proteins contained in the reagents of the in vitro expression system increased, and the expression of GFP + ERβ fusion gene and ERβ gene was increased. No specific protein band was observed in the sample subjected to the step SDS polyacrylamide gel separation, Western blot transfer, and detection by the color reaction on the streptavidin-alkaline phosphatase membrane. , GF
Approximately 80k in sample expressing P + ERβ fusion gene
About 6 in samples expressing Da and ERβ genes
A band of the expressed protein of 0 kDa could be confirmed. Similarly, in the sample in which the luciferase gene was expressed, the expected band of 61 kDa was confirmed, confirming that the in vitro expression system was working correctly (FIG. 7).

3) Purification method by antibody The in vitro synthesized protein solution obtained as described above was recovered and purified as follows. First, DE-5
Crude purification was performed by ion exchange chromatography using 2 or CM-52 cellulose. Equilibration buffer p
0.1 to 0.3 for H (3.0 to 7.0)
Purification was carried out by setting conditions under which the contaminating proteins could be separated under the MNaCl gradient elution conditions. It was observed that the estrogen receptor protein and hemoglobin dissociate favorably in the buffer solution having the pH of the peak value.
Furthermore, it was subjected to purification by affinity chromatography using a specific monoclonal antibody against the estrogen receptor. Affinity column is CN-Br
Activated Sepharose 4B (trade name, Pharmacia) was used as a carrier for affinity chromatography. Column equilibration buffer is 0.2MPB, 0.2MN
Elution was performed with 5M guanidine hydrochloride at aCl, pH 6.5.

For the estrogen receptor obtained by the above purification method, data could be obtained as one molecule of fluorescent molecule by the fluorescence correlation analysis method.

4. Environmental hormone detection system using GFP-ER by in vitro transcription By adding estradiol as a sample, the environmental hormone detection system was able to be established.

The apparatus used was a Zeiss Confocor, which was irradiated with an argon laser of 488 nm, and the fusion protein of GFP and estradiol produced by the above procedure was measured. The solution was prepared by diluting the GFP and estradiol 20 times with a phosphate buffer solution, dropping 5 μL of the solution into a Labtech chamber (Nunc #), measuring with NDF1.5 for 60 seconds, and measuring “1 molecule of data 5 times” The average value of the "fluorescence intensity per unit: COUNT / MOL" was plotted on the graph 1 shown in FIG. 8, and the standard deviation was shown as variation (FIG. 8).

As a result, with estradiol, 10
It was found that a high binding strength was exhibited at a concentration of ^-8 M, and the sensitivity was similar to that of the conventional hormone action. Furthermore, as in the case of conventional e-SCREEN using cells, a decrease in binding strength was observed at high concentrations, and it was revealed that the same signal reaction as in cells was reproduced.

5. Method of binding protein to fluorescent beads Further, even if a protein produced by an expression vector without a fluorescent label is adsorbed to the fluorescent beads, F
Analytes may be tested for specific binding using CS technology.

For example, fluorescent beads have a particle size of 100-5.
Those having a uniform particle size in the range of 00 nm and less than 3% of CV are used. Wash approximately 20 μL of the fluorescent bead suspension well with buffer by centrifugation,
On the other hand, several μg of a protein solution (for example, specific antibody or receptor) was mixed and shaken in a buffer solution at 4 ° C. for 2 hours,
Adsorb. Centrifuge to collect fluorescent beads. In order to remove floating proteins, centrifugation is repeated four times with a buffer solution to wash the fluorescent beads. Protein is adsorbed on the remaining fluorescent beads.

Using this, a substance that specifically binds to a specific antigen of an antibody or a receptor can be specifically detected by a single molecule photometric method.

[0073]

INDUSTRIAL APPLICABILITY According to the present invention, there are provided a method and an apparatus capable of accurately detecting the presence or absence of the binding ability of a test substance to a protein.

In particular, according to the present invention, a protein which maintains a three-dimensional structure which is originally obtained in a living body (that is, this protein is a biomolecule originally produced in a living body)
Can be synthesized in vitro. Furthermore, it is possible to add a fluorescent label while maintaining such a structure. This makes it possible to simulate the in vivo behavior of the target protein in vitro in a cell-free condition. That is, by allowing the test substance to act in such a state, it becomes possible to test the affinity between the target protein and the test substance. In addition, if the test is performed under such conditions, association with meaningless molecules in cells (eg, cell skeletal proteins and lipids forming various organs) which is a problem in the conventional method using FCS is caused. It is possible to avoid. Therefore, it is possible to obtain an accurate test result as compared with the conventional method. Further, it is possible to quantitatively test the affinity of the test substance for the protein.

The fusion gene of GFP and biomolecule produced according to the present invention can be stably stored. Therefore, it can be stably stored as a fusion gene for a long period of time without a change in the three-dimensional structure of the protein during the long-term storage and inactivation of its function as in the conventional method. Also,
Even when the behavior is measured by FCS, the fusion gene can be transcriptionally activated in vitro and expressed immediately after being expressed as a protein, so that the measurement can be performed, so that the change in the three-dimensional structure during the reaction and the inactivation due to the change. Can be minimized.

Further, by producing a fluorescently labeled hormone receptor according to the present invention and using it to detect the presence or absence of the binding ability of a test substance to the hormone receptor,
It is possible to easily and easily screen for the presence or absence of endocrine disrupting action of a test substance.

In particular, the estrogen receptor is localized in the nucleus. It is difficult to measure the fluctuation of molecules localized in the nucleus by the conventional method of detecting a fluorescent labeled hormone receptor in cells using FCS. On the other hand, according to the present invention, it is possible to accurately and easily measure the fluorescence-labeled hormone receptor molecule by FCS. Chemicals that bind to the estrogen receptor have been a source of ongoing pollution as a type of endocrine disrupting chemical, but can also be part of the screen for such endocrine disrupting chemicals.

[Brief description of drawings]

FIG. 1 is a diagram showing a preferred example of a fluorescence correlation analyzer.

FIG. 2 is a diagram showing a vector map relating to the vector used in one embodiment of the present invention.

FIG. 3 is an electrophoretic photograph.

FIG. 4 is an electrophoretic photograph.

FIG. 5 is an electrophoretic photograph of a fragment obtained by cleaving the GFP + ER clone with a restriction enzyme.

FIG. 6 is an electrophoretic photograph of a fragment obtained by cleaving an ER clone with a restriction enzyme.

FIG. 7 is an electrophoretic photograph of the product obtained in the in vitro expression system.

FIG. 8 is a graph showing the results of detecting endocrine disrupters using GFP-ER by in vitro transcription.

[Explanation of symbols]

1. Laser light source 2. Light intensity adjusting means 3. Optical attenuation selection device 4. Dichroic mirror 12. Photodetector 13. Fluorescence intensity recording means 14. Fluorescence intensity decay rate detection means

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12P 21/02 G01N 33/58 Z G01N 33/58 C12N 15/00 AF term (reference) 2G045 BA01 BA11 BA13 BA14 BB03 BB20 CB01 CB21 DA12 DA13 DA14 DA24 FA12 FA16 FA34 FB03 FB04 FB05 FB06 FB07 FB12 JA01 4B024 AA11 BA63 BA80 CA02 CA07 DA06 DA20 EA04 GA11 HA01 HA04 HA11 4B064. EA50 FA74 GA23 GA26

Claims (10)

[Claims] 1. A method for detecting the presence or absence of the binding ability of a test substance to a protein; (1) the presence of a protein labeled with a fluorescent substance in a solution; While continuously measuring the fluorescence intensity, the test substance is allowed to act on the labeled protein described in (1), and based on the obtained change in the fluorescence intensity over time, the protein concentration of the protein is increased. Determining the presence or absence of the binding ability of the test substance; 2. A method for detecting the presence or absence of the binding ability of a test substance to the protein according to claim 1, further comprising: (3) a change in fluorescence intensity over time obtained in (2), Determining the presence or absence of the binding ability of the test substance to the protein based on a comparison with a change in fluorescence intensity over time obtained in the absence of the test substance. 3. The detection method according to claim 1, wherein the (1) comprises: (a) a gene encoding the protein and a fluorescent substance for labeling the protein. And a promoter for expressing the gene encoding the protein extracellularly is constructed into a vector to construct an expression vector; (b) expression and transcription of the gene according to (a), and protein Producing the protein labeled with the fluorescent substance by allowing the expression vector to be present in a solution under a production-enabling condition. 4. The method according to any one of claims 1 to 3, wherein the protein is selected from the group consisting of hormone receptors, antigens and antibodies. 5. The detection method according to claim 1, wherein the protein is an estrogen receptor. 6. The detection method according to claim 3, wherein the protein is estrogen receptor β, the labeling fluorescent substance is fluorescent green protein, and the promoter is T3, T7 or SP6. A detection method, which is an expression vector selected from a promoter group. 7. The detection method according to claim 1, wherein the measurement and the determination are performed by fluorescence correlation spectroscopy. 8. A method for producing a fluorescently labeled protein, comprising: (a) a gene encoding the protein, a gene encoding a fluorescent substance for labeling the protein, and encoding the protein in a solution. Constructing an expression vector by incorporating a promoter for expressing the gene to be expressed in the vector; And producing a protein labeled with the fluorescent substance. 9. The method for producing a labeled protein according to claim 8, wherein the protein is estrogen receptor β, and the labeling fluorescent substance is fluorescent green protein,
R in which the promoter is T3, T7 or SP6
A production method, which is an expression vector selected from the NA polymerase promoter group. 10. A labeled protein produced by the production method according to claim 8 or 9.