WO2009157452A1 - Cyclic gmp detection method - Google Patents

Abstract

Disclosed is a cGMP detection method utilizing a cGMP detection system, which has wider applicability.  Specifically, the cGMP detection method comprises the steps of: introducing a polypeptide into the cGMP detection system; introducing luciferin into the cGMP detection system; and detecting a light emitted from the cGMP detection system, wherein the polypeptide comprises a domain N having a partial sequence of an N-terminal region of luciferase, a cGMP-binding domain, and a domain C having a partial sequence of a C-terminal region of luciferase in this order, wherein the domains are linked in this order, and wherein the domain N and the domain C can be bound to each other complementarily.

Description

cyclicGMP detection method

The present invention relates to a cyclic GMP (cGMP) detection method.

Eukaryotes transmit signals via second messengers to adapt to the external environment. cGMP is one of the representative second messengers, but knowing its abundance and quantitative changes in the living body is very important in analyzing information transmission of higher organisms (Beavo, JA, and LL). Brunton. 2002. Cyclic nucleotide research-still expanding after half a century. Nat Rev Mol Cell Biol. 3: 710-8.).

In higher animal cells, cGMP detection probes based on the detection principle by FRET have been developed, and it has become possible to analyze some physiological reactions involving cGMP at the cellular level (Cawley, SM, CL Sawyer). , KF Brunelle, A. van der Vliet, and WR Dostmann. 2007. Nitric oxide-evoked transient kinetics of c GMP in vascular smooth muscle cells. Cell Signal. 19: 1023-33 .; Honda, A., SR Saws , V. Lev-Ram, RY Tsien, and WR Dostmann. 2001. Spatiotemporal dynamics of guanosine 3 ', 5'-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci U S437 .; Honda, A., SR Adams, CL Sawyer, V. Lev-Ram, RY Tsien, and WR Dostmann. 2001. Spatiotemporal dynamics of guanosine 3 ', 5'-cyclic monophosphate revealed by a genetically endicator c Natl Acad Sci U S A. 98: 2437-42 .; Honda, A., MA Moosmeier, and WR Dostmann. 2005. Membrane-permeable cygnets: rapid cellular internalization of fluorescent cGMP-indicators. Front Biosci. 10: 1290-301 .; Honda, A., CL Sawyer, SM Cawley, and WR Dostmann. 2005b. Cygnets: in vivo characterization of novel cGMP indicators and in vivo imaging of intracellular cGMP. Methods Mol Biol. 307: 27-laS. , And MJ Lohse. 2006. Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat Methods. 3: 23-5 .; Sato, M., N. Hida, T. Ozawa, and Y. Umezawa. 2000. Fluorescent c on cGMP-dependent-protein kinase Ialpha and green fluorescent proteins. Anal Chem. 72: 5918-24 .; Newton, RP, and CJ Smith. 2004. Cyclic nucleotides. Phytochemistry. 65: 2423-37.). However, the analysis with the FRET probe requires that the biological sample be irradiated with excitation light from the outside, and the amount of fluorescent radiation is small, so it has an animal individual covered with skin tissue and a developed green tissue with chloroplasts. It is difficult to apply to the analysis of individual plants. As described above, in order to detect cGMP in cells in animals and plants, it is necessary to disrupt tissues or cells, and it is difficult to detect cGMP while being alive in animals and plants. For this reason, it has not been easy to analyze an information transmission system in a living body, particularly analysis of dynamic change or analysis in real time.

In view of the above problems, an object of the present invention is to provide a cGMP detection method with a wider application range.

The polypeptide of the present invention has a domain N having a partial sequence on the N-terminal side of luciferase, a cGMP binding domain, and a domain C having a partial sequence on the C-terminal side of luciferase in this order, and cGMP binds to the cGMP binding domain. When it is not, it has no luciferase activity, and has clucidase activity when cGMP binds to the cGMP binding domain. The N-terminal partial sequence and the C-terminal partial sequence may be derived from a firefly (Photinus pyralis) luciferase, a beetle (Pyrophorus plagiophthalamus) green luminescent luciferase, or a red luminescent luciferase. It may have an amino acid sequence of any combination of 2, 3 and 4, or 5 and 6. The C-terminal partial sequence is derived from a mutant of red light-emitting click luciferase in which the (420, 421, 453) amino acids (Phe, Gly, Glu) are mutated to amino acids (Ile, Ala, Ser), respectively. , The N-terminal partial sequence may be derived from firefly luciferase, green luminescent click luciferase, or red luminescent click luciferase, and the C-terminal partial sequence has the amino acid sequence of SEQ ID NO: 7, May have the amino acid sequence of SEQ ID NO: 1, 3, or 5. The cGMP binding domain may be the cGMP binding domain of phosphodiesterase 5 (PDE5), and may have the amino acid sequence of SEQ ID NO: 8.

The DNA of the present invention is characterized by encoding any of the above polypeptides. The expression vector of the present invention has this DNA and expresses any of the above polypeptides.

Further, the method of detecting cGMP in the detection system of the present invention comprises a step of introducing the expression vector into the detection system, a step of introducing luciferin into the detection system, and a step of detecting luminescence from the detection system. It is characterized by including. This detection system may be a cell.

In addition, the detection kit for detecting cGMP according to the present invention includes at least one of any of the above polypeptides or any of the above expression vectors.

== Cross-reference to related applications ==
This application claims priority based on Japanese Patent Application No. 2008-164927 filed on June 24, 2008, and is incorporated herein by reference. .

It is a schematic diagram which shows the principle of the cGMP detection in this invention. FIG. 2 is a schematic diagram showing the structures of expression vectors pCDNA4-FNFC-PDE5 and pCDNA4-GNv4m26-PDE5 used in one example of the present invention. In one Example of this invention, it is a figure which shows the result of having detected fluorescence emission by cGMP over time using the light emission detection probe GNv4m26-PDE5 in the animal cell crushed material. In one Example of this invention, it is a figure which shows the result of having detected the fluorescence emission by cGMP with respect to the density | concentration of various cGMP analogs using the luminescence detection probe FNFC-PDE5 in the animal cell crushed material. In one Example of this invention, it is a figure which shows the result of having detected the fluorescence emission by cGMP with respect to the density | concentration of various cGMP analogs using the light emission detection probe GNv4m26-PDE5 in the animal cell debris. In one Example of this invention, it is a figure which shows the result of having carried out the time-lapse observation of the light emission of the light emission detection probe accompanying cGMP detection using the light emission detection probe FNFC-PDE5 probe in the animal cell. The figure which shows the result of having measured luminescence over time using the dish type luminometer, stimulating a cell with SNP or isoproterenol using the luminescence detection probe FNFC-PDE5 probe in an animal cell in one Example of the present invention. It is. The figure which shows the result of having measured the light-emission time-lapse using the dish type luminometer, stimulating a cell with SNP or isoproterenol using the light emission detection probe GNv4m26-PDE5 probe in the animal cell in one Example of this invention. It is. In one Example of this invention, it is a figure which shows the result of having detected the increase | augmentation of the fluorescence emission by cGMP using the luminescence detection probe GNv4m26-PDE5 in the plant cell disruption with respect to the stimulation by a cGMP analog. In one Example of this invention, it is a figure which shows the result of having detected the fluorescence emission by cGMP with respect to the density | concentration of various cGMP analogs using the light emission detection probe GNv4m26-PDE5 in the plant cell disruption. In one Example of this invention, it is a figure which shows the result of having detected the fluorescence emission by cGMP with respect to cGMP produced and induced | guided | derived by the salt and the osmotic pressure stimulation using the luminescence detection probe GNv4m26-PDE5 in the plant cell disruption. In one Example of the present invention, luminescence detection probe GNv4m26-PDE5 was used in plant cell disruption, and the results of detecting fluorescence emission by cGMP over time for cGMP produced and induced by salt or osmotic pressure stimulation are shown. FIG. In one Example of this invention, it is a figure which shows the result of having carried out the time-lapse observation of the light emission of the light emission detection probe accompanying the cGMP detection in a structure | tissue.

Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. Unless otherwise stated in the embodiments and examples, J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular.cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, JG Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols A method described in a standard protocol collection such as in Molecular Biology, John Wiley & Sons Ltd., or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== cGMP detection luminescent probe ==
In general, luciferase has a domain N having a partial sequence on the N-terminal side and a domain C having a partial sequence on the C-terminal side, and these domains interact to produce luminescence activity. Here, domain N is defined as a domain having SEQ ID NO: 1 or a homologous sequence thereof in firefly luciferase, and domain C is defined as a domain having SEQ ID NO: 2 or a homologous sequence thereof in firefly luciferase.

The cGMP detection luminescent probe of the present invention is a polypeptide having a domain N, a cGMP binding domain, and a domain C in this order, and the domains N and C can be complemented. Here, that two domains derived from luciferase are complementary means that each domain does not have luminescence activity individually, but a peptide to which these domains are bound has luminescence activity. In the present specification, in particular, in a peptide in which three domains of domain N, cGMP binding domain, and domain C are bound in this order, when cGMP is not bound to the cGMP binding domain, there is no luminescence activity, and cGMP When bound to the cGMP binding domain, the structure of this peptide changes, and it means that the domain having the partial sequence of luciferase at both ends binds to produce luminescence activity. FIG. 1 shows a schematic diagram of this principle.

The partial sequence on the N-terminal side and the partial sequence on the C-terminal side may be derived from firefly luciferase, green light-emitting click luciferase, or red light-emitting click luciferase. For example, each partial sequence pair is SEQ ID NO: 1, 2, 3 4 or 5 and 6 may be used. However, as long as domain N and domain C can complement each other, the amino acid sequence of the protein shown in SEQ ID NO. That is, an amino acid sequence in which one or several amino acids are substituted, deleted, or added is permissible. In addition, domains derived from different luciferases may be paired, and in particular, the C-terminal partial sequence includes the 420th amino acid from Phe to Ile, the 421st amino acid from Gly ア ミ ノ 酸 to Ala, and the 453rd amino acid. Is derived from a mutant of red light-emitting click luciferase mutated from Glu to Ser, the domain C can be complementary to the domain N regardless of the species from which the N-terminal partial sequence is derived. In this case, for example, the partial sequence on the N-terminal side may be derived from any of firefly luciferase, green luminescent click luciferase, and red luminescent click luciferase. Specifically, the amino acid sequence of SEQ ID NO: 7 is used as the C-terminal partial sequence, and the amino acid sequence of SEQ ID NO: 1, 3, or 5 is used as the N-terminal partial sequence.

The cGMP binding domain is not particularly limited, and the binding domain of the cGMP binding protein may be used. For example, a peptide having 154 to 308th amino acid sequence (SEQ ID NO: 8) of phosphodiesterase 5 (PDE5) can be used.

Note that these three domains may be bonded directly or via a peptide or other linker.

== cGMP detection method ==
The detection method in the cGMP detection system includes a step of introducing a polypeptide serving as a cGMP detection luminescent probe into the detection system, a step of introducing luciferin into the detection system, and a step of detecting luminescence in the detection system. This detection system is not particularly limited as long as the luciferin-luciferase reaction can occur, and may be an in vitro system such as a buffer solution, a cell extract or a cell lysate, or an in vivo system called a cell. . In the case of cells, regardless of the type of cells, they are cells in the body of multicellular organisms such as animals and plants, and even if they form tissues or individuals, they are microorganisms, cultured cells, etc. May exist as

When detecting in vitro systems, cGMP detection luminescent probes can be prepared by chemical synthesis or synthesized using cells such as microorganisms or cultured cells, in vitro transcription systems or translation systems using genetic recombination technology. I do not care. In particular, when a cell extract or cell disruption is used as a detection system, an expression vector of a cGMP detection luminescent probe is introduced into the cell in advance, and the cell is extracted or disrupted after expressing the cGMP detection luminescent probe. Alternatively, after extracting or crushing cells for a detection system, a separately synthesized cGMP detection luminescent probe may be added. Thereafter, the addition of luciferin to the detection system and the luminescence measurement in the detection system may be performed in accordance with conventional methods.

When cells are used as the detection system, a transmembrane peptide such as HIV-1 TAT protein may be used to introduce the cGMP detection luminescent probe into the cells. However, from the viewpoint of ease of introduction, cGMP detection luminescence is possible. It is preferable to introduce a cGMP detection luminescent probe into a cell by introducing an expression vector that expresses the probe and expressing it in the cell. The type and configuration of this expression vector are not particularly limited as long as the DNA encoding the cGMP detection luminescent probe is inserted under an appropriate promoter and is configured to express the cGMP detection luminescent probe. The method for introducing the expression vector into the cell is not particularly limited, and injection, electroporation, lipofection, etc. may be used. In order to introduce luciferin into cells, it may be added directly to a medium or a tissue piece and allowed to stand for a while. It is also possible to introduce the cells while disrupting them by adding them together with the solubilizer. Thereafter, the luminescence measurement in the cells may be performed according to a conventional method.

The base sequence of the DNA encoding the cGMP detection luminescent probe may be a base sequence unique to each species encoding luciferase, but may be artificially determined from the amino acid sequence of the cGMP detection luminescent probe. For example, domain N (amino acid sequence is SEQ ID NO: 2) and domain C (amino acid sequence is SEQ ID NO: 2) of firefly luciferase, domain N (amino acid sequence is SEQ ID NO: 3) and domain C (amino acid sequence is SEQ ID NO: 3) of green light emitting luciferase No. 4), domain N (amino acid sequence is SEQ ID NO: 5) and domain C (amino acid sequence is SEQ ID NO: 6) of red luminescent click luciferase, and domain C (amino acid sequence is SEQ ID NO: 7) of the aforementioned red luminescent click luciferase variant The unique base sequences of the cGMP binding domain (amino acid sequence is SEQ ID NO: 8) of phosphodiesterase 5 (PDE5) are shown in SEQ ID NOs: 9 to 16, respectively. Not limited, base sequence artificially from each amino acid sequence and codon table It may be determined.

== cGMP detection kit ==
In order that anyone can easily execute the above cGMP detection method, the reagent used for the detection method may be made into a kit.
This cGMP detection kit only needs to contain the cGMP detection luminescent probe of the present invention or the DNA encoding the same, but if it contains DNA, it is included in the form of an expression vector that expresses the cGMP detection luminescent probe. It is preferable that

(1) Construction of cGMP detection luminescent probe A firefly luciferase cDNA was used as a template, the C-terminal fragment (FC) of luciferase was amplified by PCR using the following primers, cleaved with XhoI-ApaI, and then transferred to the XhoI-ApaI site of plasmid pcDNA4. This was inserted to prepare pCDNA4-FC.
FLuc-CXho-F: 5'-GTACTCGAGTGGAGGCGGCGGATGGCTACATTCTGGAGA-3 '(SEQ ID NO: 17)
FLuc-CApaI-R: 5'-GTAGGGCCCACGGCGATCTTTCCGCCCTT-3 '(SEQ ID NO: 18)

The C-terminal fragment (TC) of luciferase was synthesized by PCR using the click luciferase cDNA as a template, cleaved with XhoI-ApaI using the following primers, and inserted into the XhoI-ApaI site of plasmid pcDNA4 to produce pCDNA4-TC did.
TLuc-CXho-F: 5'-CTCGAGTGGAGGCGGCGGAAGCAAGGGTTATGTCAAT-3 '(SEQ ID NO: 19)
TLuc-CApaI-R: 5'-CCGCGGGCCCACACCGCCGGCCTTCACCAA-3 '(SEQ ID NO: 20)

By performing PCR mutagenesis on this pCDNA4-TC using the following primers, pCDNA4-m26 having a mutant (m26) of the C-terminal fragment (TC) of luciferase was prepared.
TLucC-F420I-F: 5'-CATTCTGGTGATATTGGATATTACGACGAAGATGAG-3 '(SEQ ID NO: 21)
TLucC-F420I-R: 5'-CTCATGTTCGTCGTAATATCCAATATCACCAGAATG-3 '(SEQ ID NO: 22)
TLucC-G421A-F: 5'-CATTCTGGTGATTTGCATATTACGACGAAGATGAG-3 '(SEQ ID NO: 23)
TLucC-G421A-R: 5'-CTCATCTTCGTCGTAATATGCAAAATCACCAGAATG-3 '(SEQ ID NO: 24)
TLucC-E453S-F: 5'-CCAGCTGAGTTGGAGTCGATTCTGTTGAAAAATCCATGC-3 '(SEQ ID NO: 25)
TLucC-E453S-R: 5'-GCATGGATTTTTCAACAGAATCGACTCCAACTCAGCTGG-3 '(SEQ ID NO: 26)

The N-terminal fragments (FN and GNv4) of each luciferase were amplified by PCR using the firefly and click beetle luciferase cDNA as a template, and then inserted into the HindIII-BamHI site of plasmid pcDNA4 after digestion with HindIII-BamHI. PCDNA4-FN and pCDNA4-GNv4 were prepared.
FLuc-NHindIII-F: 5'-TTTAAGCTTGCCATGGAAGACGCCAAAAACATAAAGAAAGGC-3 '(SEQ ID NO: 27)
FLuc-NBamHI-R: 5'-TTTGGATCCTCCGCCTCCTCCATCCTTGTCAATCAAGGCGTTGGT-3 '(SEQ ID NO: 28)
GNv4-NHindIII-F: 5'-TTTAAGCTTGCCATGGAGAGAGAGAAGAACGTGGTGTACGGC-3 '(SEQ ID NO: 29)
GNv4-NBamHI-R: 5'-TTTGGATCCTCCGCCTCCTCCATAGCCTTTTGT-3 '(SEQ ID NO: 30)

Insert the FC fragment and m26 fragment excised from pCDNA4-FC and pCDNA4-m26 into the XhoI-ApaI sites of plasmid pCDNA4-FN and pCDNA4-GNv4, respectively, containing the N-terminal fragments of firefly and click beetle luciferases. FNFC and pCDNA4-GNv4m26 were prepared.

Using the plasmid pSVLhPDE5 (Yanaka et al. 1998) containing human phosphodiesterase 5A (HsPDE5A) cDNA as a template, the cGMP binding domain (CGBD) was amplified by PCR using the following primers.
5'-Bgl-PDE5A: 5'-TTTAGATCTGAATTAGTGAAGGATATTTCTAGTCAT-3 '(SEQ ID NO: 31)
3'-Xho-PDE5A: 5'-AAACTCGAGTGCCAAATAAGCAGCAAAGTCCTTTTC-3 '(SEQ ID NO: 32)

The obtained CGBD fragment was cleaved with BamHI-XhoI and then inserted into the BamHI-XhoI sites of pCDNA4-FNFC and pCDNA4-GNv4m26 to obtain pCDNA4-FNFC-PDE5 and pCDNA4-GNv4m26-PDE5 (see Fig. 2). Used in.

(2) Detection of cGMP in animal cell debris In this example, animal cell debris containing a luminescent probe for cGMP detection was added to 8-Br-cGMP and 8-Br, which are membrane-permeable analogs of cGMP and cAMP. A luminescence measurement experiment was performed in which -cAMP was added to detect the luminescence of the probe.

First, 2 μg of cGMP detection luminescent probe expression vector pGNv4m26-PDE5 was introduced into HEK293 cells that were grown in 2 ml of culture medium using lipofectamin 2000 (Invitrogen). After 36 to 48 hours, the cells are collected, and 0.15 mL of a solubilizer-containing luciferin solution (Promega's Bright-Glo Luciferase Assay System) is added to homogenize the cells to obtain a cell disruption containing a cGMP detection luminescent probe. It was.

When 15 μL of 10 mM 物 8-Br-cGMP was added to 150 μL of this cell disruption and the luminescence intensity was measured with a luminometer every 90 seconds, the luminescence intensity of the cell disruption increased with time. The results of three independent experiments are averaged and shown in FIG.

Next, luminescence was measured by changing the concentration of the added cGMP analog. cGMP detection Luminescent probe expression vectors pCDNA4-FNFC-PDE5 or pCDNA4-GNv4m26-PDE5 were similarly expressed in HEK293 cells to obtain cell debris and various concentrations of 8-Br-cGMP and 8 μm between 0.1 μM and 1 mM. -Br-cAMP was added at 1.5 minutes, and the luminescence after 10.5 minutes was measured. FIG. 4 (pCDNA4-FNFC-PDE5) and FIG. 5 (pCDNA4-GNv4m26-PDE5) show changes in luminescence intensity when the intensity before stimulation was set to 1 for each vector. Both probes emitted light with high selectivity for the substrate cGMP and substrate concentration dependence, and were able to detect luminescence for 1 μM substrate.

(3) Detection of cGMP in animal cells In this example, cultured animal cells that transiently expressed a cGMP detection luminescent probe were used. First, the medium was replaced with an HBSS buffer containing 500 μM luciferin, and the culture was continued for about 30 to 60 minutes to introduce luciferin into the cells. Thereafter, 8-Br-cGMP and 8-Br-cAMP were added to the culture solution, and a luminescence measurement experiment was performed to detect the luminescence of the probe.

Specifically, as in (2), pCDNA4-FNFC-PDE5 was introduced into HEK293 cells to express the FNFC-PDE5 probe. Using probe-introduced HEK293 cells seeded in 35 mm dishes, add SNP® (Sodium® Nitroprusside), a drug that promotes cGMP synthesis, to the medium at a final concentration of 5 μM, and then perform time-lapse observation using a luminescence detector for 16 minutes and 40 seconds. went. As shown in FIG. 6A, the luminescence process (photographed every (30 seconds), exposure for 15 seconds), the luminescence in the cell mass increased with time. Furthermore, as in (2), pCDNA4-GNv4m26-PDE5 was introduced into HEK293 cells to express the GNv4m26-PDE5 probe. Using probe-introduced HEK293 cells seeded in 35 mm dishes, add SNP® (Sodium® Nitroprusside), a drug that promotes cGMP synthesis, to the medium at a final concentration of 5 μM, and then perform time-lapse observation using a luminescence detector for 16 minutes and 40 seconds. went. As shown in FIG. 6B, the light emission progressed (taken every (30 seconds), exposure for 15 seconds), the light emission in the cell mass increased with time.

Next, the cells prepared in the same manner were measured over time using a dish type luminometer while stimulating the cells with SNP multiple times. That is, as shown in FIG. 7A, when stimulation was performed 3 times with 5 μM SNP, once with 50 μM SNP, and once with 1 mM 8-Br-cGMP, the stimulation-dependent increase in luminescence and the subsequent decrease in luminescence were observed. A reversible and reproducible physiological response consisting of was detected. As shown in FIG. 7B, as a negative control, even when stimulated with 10 μM isoproterenol, an agent that promotes cAMP synthesis in cells, no significant luminescence response was shown (in the figure, at 10 μMIso). It is shown).

Moreover, even when the GNv4m26-PDE5 probe was used, the same experimental results as those obtained using the FNFC-PDE5 probe were obtained as shown in FIGS. 8A and 8B.

(4) Detection of cGMP in plant cell crushed material In this example, experiments were performed in the same manner as in (2) using Deep cells, which are cultured cells of Arabidopsis thaliana. However, the gene transfer and the transient expression of the probe were carried out by the polyethyleneglycol method after protoplasting the cells with cell wall digestion enzyme (Takeuchi, M. et al. 2000. Plant J. 23: 517-25). .).

First, 1 mM 8-Br-cGMP was added to the probe-containing deep cell disruption at 1.5 minutes, and the luminescence intensity was measured after 10.5 minutes. As shown in FIG. As in the case of the cell lysate, the level increased several tens of times before 8-Br-cGMP stimulation (in the figure, − indicates before stimulation and + indicates after stimulation).

In addition, 0.01 mM, 0.1 mM, and 1 mM 8-Br-cGMP or 8-Br-cAMP was added to the probe-containing deep cell disruption at 1.5 minutes, and the luminescence after 10.5 minutes was measured. As shown, as with animal cell debris, it had a high selectivity for cGMP and responded in a concentration dependent manner. In addition, 8-Br-cGMP at a concentration of 0.01 mM could be detected.

(5) Detection of cGMP in plant cells In this example, deep cells were used and experiments were performed in the same manner as in (3). However, the gene transfer of pCDNA4-GNv4m26-PDE5 and the transient expression of the probe were in accordance with (4), and cGMP production was induced by stimulation with salt (NaCl, KCl) or osmotic pressure (sorbitol).

In Deep cells in which the GNv4m26-PDE5 probe was transiently expressed, various stock solutions (1.5 M saline, 3 M sorbitol aqueous solution, 1.5 M potassium chloride aqueous solution) were added to one-tenth of the protoplast solution. Conditions shown (0.15M saline (shown as NaCl in the figure), 0.30M sorbitol aqueous solution (shown as Sorbitol in the figure), or 0.15M potassium chloride aqueous solution (shown as KCl in the figure) When the cells were stimulated and cGMP production was induced, a significant increase in luminescence intensity was detected compared to control water (indicated as Water in the figure) (p <0.05). .

Further, as in (2), when the change in the emission intensity over time after applying the stress was followed, the emission intensity increased with time in any stress as shown in FIG.

(6) Detection of cGMP in plant tissue In this example, a tissue fragment of onion root was used as the plant tissue, and regarding the luminescence intensity of the FNFC-PDE5 probe, time-lapse observation for 37 minutes was performed in the same manner as in (3). went. However, the expression vector was introduced into a tissue piece cut to a suitable size (several centimeters square) using a razor by the particle gun method (Arimura et al. 2004. Proc Natl Acad Sci US A 101: 7805-8.).

First, a 500 μM luciferin aqueous solution was added to an onion root tissue piece in which the FNFC-PDE5 probe was transiently expressed, and allowed to stand at 23 ° C. for about 30 to 60 minutes to introduce luciferin into the cells. Thereafter, when 10 mM 8-Br-cGMP was added to the tissue piece, a remarkable increase in luminescence intensity was detected as shown in FIG. 13A.

In addition, when 1.5M NaCl was added to induce cGMP production in the plant body, a remarkable increase in luminescence intensity was detected as shown in FIG. 13B.

These results revealed that the probe developed in this study can detect changes in intracellular cGMP concentration not only in cells but also in tissues.

The present invention can provide a cGMP detection method with a wider application range.

Claims (12)

1. A domain N having a partial sequence on the N-terminal side of luciferase, a domain CGMP binding domain, and a domain C having a partial sequence on the C-terminal side of luciferase in this order,
   A polypeptide having no luciferase activity when cGMP is not bound to a cGMP binding domain and having luciferase activity when cGMP is bound to a cGMP binding domain.
2. The polypeptide according to claim 1, wherein the partial sequence on the N-terminal side and the partial sequence on the C-terminal side are derived from firefly luciferase, green light-emitting click luciferase, or red light-emitting click luciferase.
3. 2. The N-terminal partial sequence and the C-terminal partial sequence each include an amino acid sequence of any combination of SEQ ID NOs: 1, 2, 3, and 4, or 5 and 6. The described polypeptide.
4. The C-terminal partial sequence is derived from a mutant of red light-emitting click luciferase in which the 420th amino acid is mutated from Phe to Ile, the 421st amino acid is mutated from Gly to Ala, and the 453rd amino acid is mutated from Glu to Ser. The polypeptide according to claim 1.
5. The C-terminal partial sequence includes the amino acid sequence of SEQ ID NO: 7, and the N-terminal partial sequence includes any of the amino acid sequences of SEQ ID NOS: 1, 3, or 5. Item 5. The polypeptide according to Item 4.
6. The polypeptide according to any one of claims 1 to 3, wherein the cGMP binding domain contains a cGMP binding domain of phosphodiesterase 5 (PDE5).
7. The polypeptide according to claim 6, wherein the cGMP binding domain of the phosphodiesterase 5 (PDE5) is the amino acid sequence set forth in SEQ ID NO: 8.
8. DNA encoding the polypeptide according to any one of claims 1 to 7.
9. An expression vector comprising the DNA according to claim 8 and expressing the polypeptide according to any one of claims 1 to 7.
10. A method for detecting cGMP in a detection system,
    Introducing the expression vector according to claim 9 into the detection system;
    Introducing luciferin into the detection system;
    Detecting light emission from the detection system;
    A method comprising the steps of:
11. The method according to claim 10, wherein the detection system is a cell.
12. a detection kit for detecting cGMP,
    A detection kit comprising at least one of the polypeptide according to any one of claims 1 to 7 or the expression vector according to claim 8.

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