JP2009186357A - Reagent for analyzing protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for sensitively and easily measuring specific protein. <P>SOLUTION: This fluorescence reagent used for detection or quantitative determination of protein has a general formula (I): A-S<SB>1</SB>-B (where, A is fluorescence compound containing a fluorophore having hydrophobic aromatic group or hydrophobic heterocyclic group, B is peptide made of 10-60 amino acid bondable to target protein, and S<SB>1</SB>is spacer). A protein detecting method used for the fluorescence reagent is provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention is directed to general formula (I):
_A-S 1 -B (I)
[Where:
A represents a fluorescent compound containing a fluorophore having a hydrophobic aromatic group or heterocyclic group,
B represents a peptide consisting of 10 to 60 amino acids capable of binding to the target protein,
S ₁ represents a spacer. ]
The present invention relates to a fluorescent reagent for use in protein detection or quantification, and a protein detection method using the fluorescent reagent.

  In recent years, structural determination of proteins that are indicators of diseases such as cancer marker proteins has progressed, and attempts have been widely made to detect diseases by qualitative analysis or quantitative analysis of such proteins. In particular, research on molecules related to angiogenesis closely related to cancer growth and metastasis has progressed, and it has become clear that molecules such as VEGF (vascular endothelial growth factor), ephrin, and angiopoietin are deeply involved in cancer pathology. It has become. In this context, VEGF is of particular interest, and there is a need for methods for high-throughput analysis, including qualitative and quantitative analysis of the molecule.

  On the other hand, with the development of DNA chip technology, transcriptome analysis for comprehensive analysis of RNA expression has been actively performed. However, the RNA expression profile obtained by such a technique and the protein expression profile do not always coincide with each other, and at present, the correlation is estimated to be 50% or less. Therefore, it is considered important not only to perform comprehensive analysis of RNA but also to analyze protein expression itself.

  Regarding protein expression, two-dimensional electrophoresis and mass spectrometry have been remarkably developed, and high-throughput analysis methods comparable to nucleic acid analysis have appeared, such as protein chips. In the future, in order to utilize the results of such technological development in the field of medical care and health management, it is necessary to establish a simple and rapid analysis technique for proteins that can be disease markers such as VEGF. ing.

  Representative methods for quantifying proteins in a sample include (1) absorptiometry, (2) Biuret method using Biuret reagent, (3) Lowry method combining phenol reagent and Biuret method, (4) Bradford method etc. are mentioned. As a method for specifically measuring a specific protein, (5) ELISA is used. The characteristics of these methods are as follows.

(1) Spectrophotometric method: a method for calculating a protein concentration using absorption near 280 nm caused by tyrosine and tryptophan contained in a protein. Since the tyrosine and tryptophan contents differ depending on the type of protein, the absorbance at 280 nm (A280 nm) can vary even at the same concentration. Therefore, A280 nm at a control concentration (such as 1 mg / mL) is usually 1. As 0, the protein concentration in the sample is calculated. The operation is simple and the sample can be collected after the measurement. A280 nm varies depending on the type of protein. Measurement is not possible for proteins that do not absorb light near 280 nm (eg, collagen, gelatin). Quantification is hindered by contamination with substances that absorb ultraviolet light.

(2) Biuret method: a protein is reacted with a Cu ²⁺ solution under alkaline conditions, and the red color exhibited by coordination of Cu ²⁺ with a nitrogen atom in a polypeptide chain is measured from the absorbance at 540 nm. This is a method for calculating the concentration. There is little difference in color development rate depending on the type of protein. Easy to operate. The sensitivity is low. Therefore, it cannot be used for the measurement of a low concentration sample.

(3) Lowry method: The blue color produced by reacting a protein with a phenol reagent (phosphomolybdic acid and phosphotungstic acid dissolved in an acidic solution) and reacting the phenol reagent with tyrosine, tryptophan and cysteine in the protein. It is a method of measuring the protein concentration by detection. Due to the strong color development derived from peptide bonds, it is much more sensitive than the Biret method and is the most commonly used method. Since coloration occurs due to the reduction reaction, it is interfered with by other reducing substances. The operation is complicated and takes time to measure. The color development rate varies depending on the type of protein.

(4) Bradford method: Color change caused by binding of Coomassie Brilliant Blue G-250 (triphenylmethane blue dye) to protein in acidic solution (red purple to blue, maximum absorption wavelength: shifted from 465 nm to 595 nm) ) To quantify the protein. Less susceptible to contamination. Easy to operate. The color development rate varies depending on the type of protein. Color development is inhibited by the incorporation of a surfactant.

(5) ELISA method: An immunological method for quantifying a protein of interest using an antibody that specifically binds to a specific protein. The concentration of a particular protein can be measured. It takes time to measure.

  In view of the various shortcomings that exist in these techniques, it will be appreciated that new techniques are needed to detect and measure proteins.

  Here, a fluorescence measurement method is used for measuring various chemical substances. The fluorescence measurement method has advantages such as high sensitivity, a small amount of sample required, and no need for a large-scale apparatus or skilled technique.

  Protein quantification using a fluorescence measurement method has been reported so far. As a method using a direct bond between a fluorescent dye and a protein, for example, a method using fluorescamine (utilizing that when fluorescamine binds to a primary amine, it becomes a fluorescent substance emitting strong fluorescence at 495 nm), There are a method using a cyanine dye and a method using an azole derivative (Patent Document 1). However, in these methods, there are problems such that the detection rate varies depending on the type of protein, the calibration curve does not become a straight line, a measurement error occurs due to association of dyes, and the Stokes shift is small. Furthermore, these proteins detect all proteins nonspecifically. As a method for specifically detecting a specific protein using fluorescence measurement, a method using a labeled probe peptide (Patent Document 2), a method using a labeled antibody (Patent Document 3), and the like have been developed. .

Japanese Patent Laid-Open No. 2006-234772 Japanese Patent Laid-Open No. 2002-14102 JP 2004-77387 A

  Then, an object of this invention is to provide the means which can measure a specific protein highly sensitively and simply.

  The present inventors have found that the fluorescent reagent of the present invention in which a peptide capable of specifically binding to a target protein is linked to a fluorescent compound does not produce significant fluorescence in the absence of the target protein, but acts on the target protein. Then, the peptide portion in the fluorescent reagent binds to the target protein, so that the fluorescent compound interacts with the target protein and the vicinity of the fluorophore of the fluorescent compound becomes a hydrophobic environment, thereby generating fluorescence from the fluorescent compound. As a result, the present invention has been completed.

That is, the present invention has the following features.
(1) General formula (I):
_A-S 1 -B (I)
[Where:
A represents a fluorescent compound containing a fluorophore having a hydrophobic aromatic group or heterocyclic group,
B represents a peptide consisting of 10 to 60 amino acids capable of binding to the target protein,
S ₁ represents a spacer. ]
A fluorescent reagent for protein analysis, wherein fluorescence is generated when the target protein bound to B interacts with the hydrophobic group of A.

(2) It further has a group C for binding to the solid phase, and has the general formula (II):
_{A-S 1 -B-S 2} -C (II)
[Where:
A represents a fluorescent compound containing a fluorophore having a hydrophobic aromatic group or heterocyclic group,
B represents a peptide consisting of 10 to 60 amino acids capable of binding to the target protein,
C represents a moiety that binds to the solid phase;
S ₁ and S ₂ represent spacers. ]
The fluorescent reagent according to (1) above, represented by:

(3) The fluorescent reagent according to the above (1) or (2), wherein the spacer S ₁ and / or S ₂ is a substituted or unsubstituted alkylene, oxyalkylene or peptide.

(4) The hydrophobic aromatic group or heterocyclic group of the fluorescent compound A is substituted or unsubstituted naphthyl, anthranyl, pyrenyl, biphenyl, coumarin, dansyl, benzothiazolyl, fluorescein, rhodamine, pyridine, bipyridine, quinolyl and The fluorescent reagent according to any one of (1) to (3) above, which is a group selected from the group consisting of phenanthroyl.

(5) The fluorescent reagent according to any one of (1) to (4), wherein the fluorescent compound A is fluorescein, dansyl, rhodamine or a fluorescent derivative thereof.
(6) The fluorescent reagent according to any one of (1) to (5), wherein the target protein capable of binding to the peptide B is VEGF.
(7) The fluorescent reagent according to (6) above, wherein the peptide B comprises the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

(8) The fluorescent reagent according to the above (7), wherein in the general formula (II), the portion corresponding to S ₁ -BS ₂ -C is a peptide having the amino acid sequence shown in SEQ ID NO: 3.
(9) The fluorescent reagent according to any one of (2) to (8), wherein the group C is a group having a primary amino group, a thiol group, a carboxyl group, a hydroxyl group, or an epoxy group.
(10) An immobilized fluorescent reagent comprising the fluorescent reagent according to any one of (1) to (9) above on a solid phase.
(11) A protein detection method using the fluorescent reagent according to any one of (1) to (9) above.

  According to the present invention, highly sensitive detection and measurement of a specific protein can be realized efficiently. Furthermore, by using the present invention for high-throughput analysis of disease-related gene expression, it can be used for diagnosis, prevention and treatment of diseases.

  In the present invention, “fluorescence” means that when a certain molecule is irradiated with light of a specific wavelength (excitation light), light having a wavelength different from the irradiation light emitted from the molecule or such light is emitted. To do. In the present invention, terms such as “fluorescent compound”, “fluorescent molecule”, and “fluorescent dye” are used interchangeably, and all mean compounds capable of emitting fluorescence under specific conditions.

  In the present invention, “capable of binding” to a target protein for a peptide means that the peptide can bind to the target protein in some manner, including covalent and ionic bonds, hydrophobic bonds, van der Waals. Includes non-covalent bonds such as force bonds. The binding is preferably specific. In this case, “specific” means that the peptide binds to the target protein, but does not substantially bind to other proteins. Molecules that specifically bind to the target protein preferably bind to the target protein at a strength of 100 times or more, 1000 times or more, or 10,000 times or more compared to the binding to other proteins.

  In the present invention, the target protein is a protein capable of protein-protein interaction, such as a receptor protein having a ligand capable of binding, an adhesion protein, an antigen or an antibody. The target protein may be associated with a disease, for example. Such include proteins that can be used to diagnose a disease, such as proteins whose increase or decrease in the amount suggests the presence of the disease. Target proteins include growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), brain-derived growth factor (BDGF), vascular endothelial growth factor (VEGF), and cell adhesion factors such as fibronectin, laminin, and vitronectin. , Insulin, somatostatin, somatothrombin, hormones such as gonadotropin releasing factor, lipoproteins such as LDL, various tumor markers, antibodies and the like. In an embodiment of the invention, the target protein is vascular endothelial growth factor (VEGF).

  In the fluorescent reagent of the present invention represented by the general formula (I) or (II), A is any fluorescent compound containing a fluorophore having a hydrophobic aromatic group or heterocyclic group. Examples of molecules that can be used as A include naphthalene compounds, anthracene compounds, pyrene compounds, biphenyl compounds, coumarin compounds, benzothiazole compounds, bipyridine compounds, quinoline compounds, and phenanthroline compounds. Among aromatic / heterocyclic compounds, those that emit fluorescence are listed. Commonly used fluorescent compounds such as fluorescein, rhodamine, dansyl, Alexa, FITC, Cy3 and the like can also be used as A. Preferred fluorescent compounds are fluorescein, rhodamine, dansyl, or fluorescent derivatives thereof.

  In the fluorescent reagent of the present invention, B is a peptide that can bind to the target protein. For example, a peptide has a sequence derived from a binding domain with a target protein. The length of the peptide is preferably 10 to 60 amino acids, more preferably 15 to 40 amino acids. Various sequences of peptides capable of specifically binding to a specific protein are known, and those skilled in the art can appropriately identify such peptides. For example, when the target protein is VEGF, the amino acid sequence of the peptide that can be used as B is RGWVEICAADDYGRCL (SEQ ID NO: 1) or GNEDIARMWEWEFERRL (SEQ ID NO: 2), or 80% or more, preferably 90% of these amino acid sequences. A sequence having at least% identity. As B, an Fab domain of an antibody that specifically binds to a target protein can also be used.

  In the fluorescent reagent of the present invention, C is not particularly limited as long as it is a group that can be used for binding the fusion molecule to a solid phase. Preferred group C is a group having a primary amino group, a thiol group, a carboxyl group, a hydroxyl group, or an epoxy group. The compound having such a functional group is bound to the surface of the solid support activated using the corresponding binding group, and thus the fluorescent reagent of the present invention can be immobilized on the solid phase. For example, a group that can be bonded to an amino group is a formyl group, an ester group, an activated BrCN group, or the like, a group that can be bonded to a thiol group is a disulfide group, and the group that can be bonded to a carboxyl group is a hydroxyl group or an amino group Examples of the group that can be bonded to the epoxy group include a hydroxyl group and an amino group.

In the fluorescent reagent of the present invention, S ₁ and S ₂ are spacers having a role of linking AB and BC, respectively. Suitably the spacer is a substituted or unsubstituted alkylene, oxyalkylene or peptide, preferably a linker peptide comprising glycine. For example, the spacer is a peptide having the sequence GPGSG.

  The peptide portion of the fluorescent reagent of the present invention may be chemically synthesized or purified after being recombinantly expressed by a host cell.

  The solid phase for immobilizing the fluorescent reagent of the present invention is not particularly limited as long as it can measure fluorescence generated by binding of the target protein on the solid phase. The solid phase material can be a glass surface deposited with a thin film of glass, plastic, gold or silver. Preferred examples of the solid phase include a glass slide, a plastic plate, and a gold substrate for surface plasmon resonance analysis. However, the shape and material of the solid phase are not particularly limited.

The present invention also includes an immobilized fluorescent reagent containing the above-described fluorescent reagent on a solid phase.
Regarding the fluorescent reagent of the present invention, the fluorescent compound A, spacer S ₁ , protein-binding peptide B, spacer S ₂ and group C may be covalently bonded or non-covalently bonded. When the spacers S ₁ and S ₂ are peptides, a gap between them and peptide B is preferably a peptide bond. The bond between Compound C and the solid phase may be a covalent bond or a non-covalent bond.

  The fluorescent reagent of the present invention utilizes the fact that the fluorescence intensity of the fluorescent compound increases in a hydrophobic environment. That is, the present invention is not bound by theory, but is considered to have the following mechanism: A fluorescent reagent in which a peptide capable of binding to a target protein is linked to a fluorescent compound produces remarkable fluorescence in the absence of the target protein. Absent. However, when this is allowed to act on the target protein, the peptide moiety in the fluorescent reagent binds to the target protein, so that the fluorescent compound interacts with the target protein and the vicinity of the fluorophore of the fluorescent compound becomes a hydrophobic environment, thereby Fluorescence is generated from the fluorescent compound. An outline of the mechanism of the present invention is shown in FIG. In this case, the interaction refers to a state in which some physical and / or chemical action occurs between the target protein and the fluorescent compound. For example, the target protein has an ionic bond, hydrophobic bond, This includes non-covalent bonds such as by force.

  Any fluorescent compound can be used in the fluorescent reagent of the present invention. Moreover, the peptide which can be specifically couple | bonded with a target protein can be used as a peptide part. Therefore, detection or quantification of the target protein of the present invention can be performed using a fluorescent compound that has a sufficiently large Stokes shift, a sufficiently strong fluorescence intensity, and is easy to detect, and that is specific to the target protein. This is advantageous in that it can be performed compared to protein detection by existing fluorescence measurement methods.

  Protein detection or measurement using the fluorescence of the present invention can be performed using commercially available and commonly used equipment. For example, examples of equipment that can be used include a microplate reader, a fluorescence spectrophotometer, and a fluorescence microscope.

  As described above, the fluorescence measurement has high detection sensitivity, requires only a very small amount of sample, and can be performed by a general instrument. Therefore, very efficient protein measurement can be realized by using the fluorescent reagent of the present invention, which is applied to specific detection of a specific protein.

  The present invention includes a protein detection method using the fluorescent reagent of the present invention. Protein detection may be performed in a liquid phase, or may be performed on a solid phase by immobilizing a fluorescent reagent on the solid phase. The detection method of the present invention includes, for example, a step of bringing the fluorescent reagent of the present invention into contact with a test sample, a step of detecting / measuring fluorescence from the fluorescent reagent of the present invention, and comparing the obtained fluorescence intensity with an appropriate control. To determine the presence or concentration of the target protein in the sample. Suitable controls include fluorescence intensity when contacted with a control sample that is known not to contain the target protein, or fluorescence intensity obtained using a standard sample containing a known concentration of the target protein. It is done.

Example 1: Fluorescent reagent using fluorescein
(1) Preparation of fluorescent reagent Fluorescein is used as the fluorescent compound of A in the fluorescent reagent of the present invention, VEGF binding peptide (SEQ ID NO: 1) is used as the protein binding peptide of B, and an amino group is used as the solid phase binding group of C. using lysine with, and with the peptide having the sequence of each GPGSG and GPGS as a spacer _{S 1} and _{S 2.} Such a fluorescent reagent was synthesized using an automatic peptide synthesizer. The structure of the fluorescent reagent is shown below.

Fluorescein-GPGSG-RGWVEICAADDYGRCL-GPGS-K
A chromatogram of the fluorescent reagent in HPLC is shown in FIG. Chromatographic conditions are as follows:
Column: Supelco Discovery HS C18 4.6mm ID x 7.5cm Particle 3μm
Pressure: 7.6 MPa
Column temperature: 27.4 ° C
Eluent: 0.1% TFA
A mass spectrum of the fluorescent reagent is shown in FIG.

(2) Fluorescence spectrum measurement With respect to the fluorescence reagent prepared in (1) above, fluorescence spectrum measurement was performed in order to confirm changes in fluorescence intensity due to the presence / absence of VEGF. Human VEGF121 was used as VEGF. VEGF was obtained from PERPROTECH. The fluorescence spectrum measurement was performed using a fluorescence spectrophotometer (manufactured by JASCO Corporation). The measurement conditions are as follows:
Fluorescent reagent: 10 μM (in phosphate buffered saline)
VEGF121: 0 to 500 ng / mL
Measurement temperature: 25 ° C
Excitation wavelength: 494 nm

  The results of fluorescence spectrum measurement in the presence and absence of VEGF are shown in FIG. The VEGF concentration in the presence of VEGF is 500 ng / mL. It can be seen that the fluorescence intensity increased significantly with the addition of VEGF.

  The result of plotting the fluorescence intensity at 530 nm against the VEGF concentration is shown in FIG. It can be seen that there is a linear correlation between fluorescence intensity and VEGF concentration. This indicates that the fluorescent reagent of the present invention is useful for quantification of the target protein.

(3) Immobilization of fluorescent reagent on solid phase First, a monomolecular film coating was applied on a gold substrate using the following two types of reagents.

  Reagent 1 and reagent 2 were obtained from DOJINDO. A gold substrate (manufactured by GE Healthcare) was immersed in 2 mL of a mixed solution (in ethanol) of reagent 1 (1.9 mM) and reagent 2 (0.1 mM), and left at room temperature for 12 hours. The substrate was washed with ethanol and then dried for 30 minutes under an Ar stream. Thereafter, the substrate was mounted on a Biacore T100 sensor chip.

  The fluorescent reagent was immobilized on the gold substrate by a covalent bond between the carboxyl group of the reagent 2 constituting the monomolecular film and the amino group of the lysine residue corresponding to the group C of the fluorescent reagent of the present invention. First, the reaction activation of the carboxyl group of Reagent 2 was performed with EDC / NHS (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysuccinimide). Subsequently, the reaction-activated gold substrate was brought into contact with the fluorescent reagent of the present invention, and the fluorescent reagent was immobilized on the gold substrate. Finally, ethanolamine was added to block unreacted carboxyl groups.

The immobilization reaction was monitored by surface plasmon resonance. The reaction conditions are as follows:
EDC: 1.0 mM (in H ₂ O)
NHS: 1.0 mM (in H ₂ O)
Fluorescent reagent: 100 μg / mL (in acetate buffer (10 mM, pH 4.0))
Ethanolamine: 1.0M (pH 8.5)
Flow solution: phosphate buffered saline (0.01 M, pH 7.4)
Flow rate: 5.0 μL / min Analyzer: Biacore T100
The result of monitoring is shown in FIG.

(4) Binding between immobilized fluorescent reagent of the present invention and VEGF The binding between the fluorescent reagent immobilized on the solid phase in (3) above and VEGF 121 was monitored using surface plasmon resonance. The reaction conditions are as follows:
VEGF121: 5.0, 2.5, 1.2, 0.7 or 0 μg / mL (in phosphate buffered saline (0.01 M, pH 7.4))
Flow rate: 30 μL / min Analyzer: Biacore T100
The results are shown in FIG. The signal intensity increases as the concentration of VEGF increases.

(5) Measurement of fluorescence spectrum of immobilized fluorescent reagent of the present invention The fluorescence spectrum of the fluorescent reagent of the present invention immobilized on the substrate in (3) above was measured in the presence of various concentrations of VEGF. The measurement conditions were as follows:
VEGF: 0, 0.15, 0.30, 0.50, 1.00 μg / mL (in phosphate buffered saline (pH 7.4))
Excitation wavelength: 470 nm

  The results are shown in FIG. It can be seen that the fluorescence intensity of the fluorescent reagent of the present invention increases as the VEGF concentration increases. This indicates that the fluorescent reagent of the present invention immobilized on a solid phase is useful for quantification of the target protein.

(6) Increase in fluorescence intensity due to binding to VEGF in serum Regarding the fluorescent reagent of the present invention, increase in fluorescence intensity due to the addition of serum added with VEGF was confirmed. To the fluorescent reagent of the present invention immobilized on the substrate in the above (3), rat serum (Nippon Biotest Laboratories, Inc.) without VEGF (0 μg / mL) or VEGF added (0.2 μg / mL) is added. The fluorescence spectrum was measured. The excitation wavelength was 470 nm.

  The results are shown in FIG. It can be seen that the fluorescence intensity is remarkably high in the case of serum added with VEGF. This indicates that the fluorescent reagent of the present invention is not affected by an inhibitor even in measurement of a biological sample such as serum, and thus is useful for analysis of a target protein in such a biological sample. .

Example 2: Fluorescent reagent using dansyl
(1) Preparation of fluorescent reagent A fluorescent reagent using dansyl as the fluorescent compound A in the fluorescent reagent of the present invention was prepared in the same manner as in Example 1. The structure of the fluorescent reagent is shown below.
Dansil-GPGSG-RGWVEICAADDYGRCL-GPGS-K

(2) Measurement of fluorescence spectrum The increase in fluorescence intensity of the dansyl fluorescence reagent depending on the concentration of VEGF was measured by fluorescence spectrum measurement. The measurement conditions are as follows:
Fluorescent reagent: 10 μM (in phosphate buffered saline)
VEGF121: 0-1 μg / mL
Measurement temperature: 25 ° C
Excitation wavelength: 350 nm

  The fluorescence spectrum is shown in FIG. It can be seen that the fluorescence intensity of dansyl increases with increasing VEGF concentration.

  FIG. 11 shows the result of plotting the fluorescence intensity at 510 nm against the VEGF concentration. It can be seen that there is a linear correlation between fluorescence intensity and VEGF concentration.

  According to the present invention, a high-sensitivity and high-throughput means for quantifying a specific protein can be realized. Further, such means provides an efficient method for detecting and diagnosing diseases.

It is a figure showing the outline of fluorescence generation | occurrence | production by the coupling | bonding of the fluorescent reagent of this invention, and a target protein. It is a chromatogram of the fluorescent reagent of this invention in HPLC. It is a mass spectrum of the fluorescent reagent of the present invention. It is a fluorescence spectrum of the fluorescent reagent of the present invention before and after the addition of VEGF. It is a plot with respect to VEGF of the fluorescence intensity in 530 nm of the fluorescent reagent of this invention when mixed with VEGF. It is a result of the surface plasmon resonance showing the immobilization reaction on the board | substrate of the fluorescent reagent of this invention. It is the result of the surface plasmon resonance showing the coupling | bonding of the fluorescent reagent of this invention fixed on the board | substrate, and VEGF. It is a fluorescence spectrum of the fluorescent reagent of the present invention immobilized on a substrate. It is a fluorescence spectrum when VEGF-containing serum is added to the fluorescent reagent of the present invention immobilized on a substrate. 2 is a fluorescence spectrum of the dansyl fluorescent reagent of the present invention at various VEGF concentrations. It is a plot with respect to VEGF of the fluorescence intensity in 510 nm of the dansyl fluorescence reagent of this invention when mixed with VEGF.

SEQ ID NOs: 1-3: VEGF binding peptide

Claims (11)

Formula (I):
_A-S 1 -B (I)
[Where:
A represents a fluorescent compound containing a fluorophore having a hydrophobic aromatic group or heterocyclic group,
B represents a peptide consisting of 10 to 60 amino acids capable of binding to the target protein,
S ₁ represents a spacer. ]
A fluorescent reagent for protein analysis, wherein fluorescence is generated when the target protein bound to B interacts with the hydrophobic group of A. It further has a group C for binding to the solid phase and has the general formula (II):
_{A-S 1 -B-S 2} -C (II)
[Where:
A represents a fluorescent compound containing a fluorophore having a hydrophobic aromatic group or heterocyclic group,
B represents a peptide consisting of 10 to 60 amino acids capable of binding to the target protein,
C represents a moiety that binds to the solid phase;
S ₁ and S ₂ represent spacers. ]
The fluorescent reagent according to claim 1, represented by: The spacer S ₁ and / or S ₂ is a substituted or unsubstituted alkylene, oxyalkylene or peptide, a fluorescent reagent according to claim 1 or 2.   The hydrophobic aromatic group or heterocyclic group of the fluorescent compound A is composed of substituted or unsubstituted naphthyl, anthranyl, pyrenyl, biphenyl, coumarin, dansyl, benzothiazolyl, fluorescein, rhodamine, pyridine, bipyridine, quinolyl and phenanthryl. The fluorescent reagent according to any one of claims 1 to 3, which is a group selected from the group.   The fluorescent reagent according to any one of claims 1 to 4, wherein the fluorescent compound A is fluorescein, dansyl, rhodamine or a fluorescent derivative thereof.   The fluorescent reagent according to any one of claims 1 to 5, wherein the target protein capable of binding to the peptide B is VEGF.   The fluorescent reagent according to claim 6, wherein the peptide B comprises an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. Wherein in formula (II), it is a peptide having the amino acid sequence shown in S 1 _-B-S 2 _-C portion corresponding to SEQ ID NO: 3, a fluorescent reagent according to claim 7.   The fluorescent reagent according to any one of claims 2 to 8, wherein the group C is a group having a primary amino group, a thiol group, a carboxyl group, a hydroxyl group, or an epoxy group.   An immobilized fluorescent reagent comprising the fluorescent reagent according to any one of claims 1 to 9 on a solid phase.   A protein detection method using the fluorescent reagent according to claim 1.

Images