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WO2009157452A1 - Procédé de détection du gmp cyclique - Google Patents

Procédé de détection du gmp cyclique Download PDF

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Publication number
WO2009157452A1
WO2009157452A1 PCT/JP2009/061423 JP2009061423W WO2009157452A1 WO 2009157452 A1 WO2009157452 A1 WO 2009157452A1 JP 2009061423 W JP2009061423 W JP 2009061423W WO 2009157452 A1 WO2009157452 A1 WO 2009157452A1
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WO
WIPO (PCT)
Prior art keywords
cgmp
luciferase
domain
partial sequence
amino acid
Prior art date
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Ceased
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PCT/JP2009/061423
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English (en)
Japanese (ja)
Inventor
岳昌 小澤
雅宜 竹内
研二 三浦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Probex
PROBEX Inc
Probex Inc
University of Tokyo NUC
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Probex
PROBEX Inc
Probex Inc
University of Tokyo NUC
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Publication date
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Publication of WO2009157452A1 publication Critical patent/WO2009157452A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the present invention relates to a cyclic GMP (cGMP) detection method.
  • cGMP cyclic GMP
  • 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.).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • it is a figure which shows the result of having detected the fluorescence emission by cGMP with respect to the density
  • 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
  • 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
  • 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.
  • 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.
  • domain N is defined as a domain having SEQ ID NO: 1 or a homologous sequence thereof in firefly luciferase
  • 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.
  • 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.
  • 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.
  • each partial sequence pair is SEQ ID NO: 1, 2, 3 4 or 5 and 6 may be used.
  • 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.
  • 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 ⁇ ⁇ ⁇ ⁇ to Ala, and the 453rd amino acid.
  • the domain C can be complementary to the domain N regardless of the species from which the N-terminal partial sequence is derived.
  • 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.
  • the amino acid sequence of SEQ ID NO: 7 is used as the C-terminal partial sequence
  • 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.
  • a peptide having 154 to 308th amino acid sequence (SEQ ID NO: 8) of phosphodiesterase 5 (PDE5) can be used.
  • 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. .
  • 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
  • cGMP detection luminescent probes 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.
  • 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.
  • 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.
  • a transmembrane peptide such as HIV-1 TAT protein may be used to introduce the cGMP detection luminescent probe into the cells.
  • 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.
  • luciferin 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.
  • domain N amino acid sequence is SEQ ID NO: 2
  • domain C amino acid sequence is SEQ ID NO: 2 of firefly luciferase
  • domain N amino acid sequence is SEQ ID NO: 3
  • domain C amino acid sequence is SEQ ID NO: 3 of green light emitting luciferase No.
  • 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
  • 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.
  • 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
  • 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)
  • TC C-terminal fragment 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)
  • 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'-CCAGCTGAGTTGGAGTCGATTCTGTTGAAAAATCCAT
  • 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)
  • 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.
  • CGBD cGMP binding domain
  • 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.
  • 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.
  • a solubilizer-containing luciferin solution Promega's Bright-Glo Luciferase Assay System
  • 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 ⁇ m between 0.1 ⁇ M 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 ⁇ M substrate.
  • pCDNA4-FNFC-PDE5 was introduced into HEK293 cells to express the FNFC-PDE5 probe.
  • SNP® sodium® Nitroprusside
  • a drug that promotes cGMP synthesis was added to the medium at a final concentration of 5 ⁇ M, and then perform time-lapse observation using a luminescence detector for 16 minutes and 40 seconds. went.
  • FIG. 6A the luminescence process (photographed every (30 seconds), exposure for 15 seconds), the luminescence in the cell mass increased with time.
  • pCDNA4-GNv4m26-PDE5 was introduced into HEK293 cells to express the GNv4m26-PDE5 probe.
  • SNP® sodium® Nitroprusside
  • a drug that promotes cGMP synthesis was added to the medium at a final concentration of 5 ⁇ M, and then perform time-lapse observation using a luminescence detector for 16 minutes and 40 seconds. went.
  • FIG. 6B the light emission progressed (taken every (30 seconds), exposure for 15 seconds), the light emission in the cell mass increased with time.
  • 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 ⁇ M SNP, once with 50 ⁇ M 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 ⁇ M isoproterenol, an agent that promotes cAMP synthesis in cells, no significant luminescence response was shown (in the figure, at 10 ⁇ MIso). It is shown).
  • cGMP production was induced by stimulation with salt (NaCl, KCl) or osmotic pressure (sorbitol).
  • cGMP cGMP in plant tissue
  • a tissue fragment of onion root was used as the plant tissue
  • time-lapse observation for 37 minutes was performed in the same manner as in (3). went.
  • 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.).
  • a 500 ⁇ M 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.
  • the present invention can provide a cGMP detection method with a wider application range.

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Abstract

La présente invention concerne un procédé de détection du cGMP utilisant un système de détection du cGMP, qui possède une applicabilité plus large. Spécifiquement, l'invention concerne le procédé de détection du cGMP comprenant les étapes consistant à : introduire un polypeptide à l'intérieur du système de détection du cGMP; introduire la luciférine à l'intérieur du système de détection du cGMP; et détecter une lumière émise du système de détection du cGMP, le polypeptide comprenant un domaine N ayant une partie de la séquence d'une région N-terminale de la luciférase, un domaine de liaison au cGMP, et un domaine C ayant une partie de la séquence d'une région C-terminale de la luciférase dans cet ordre, les domaines étant liés dans cet ordre, et le domaine N et le domaine C pouvant être liés l'un à l'autre de manière complémentaire.
PCT/JP2009/061423 2008-06-24 2009-06-23 Procédé de détection du gmp cyclique Ceased WO2009157452A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011195547A (ja) * 2010-03-23 2011-10-06 Olympus Corp サイクリックｇｍｐ解析方法
EP2436764A4 (fr) * 2009-05-29 2012-11-28 Univ Tokyo Procédé de détection ultrasensible d'une interaction protéine-protéine

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000071668A2 (fr) * 1999-05-20 2000-11-30 The Board Of Regents Of The University Of Nebraska Procede d'identification de composants impliques dans les mecanismes de transduction de signaux dans les plantes superieures
JP2003509051A (ja) * 1999-09-15 2003-03-11 プロメガ コーポレイション フォツリス・ペンシルバニカ及びピロフィルス・プラギオフタラムス由来の熱安定性ルシフェラーゼ及び製造方法
JP2005245467A (ja) * 1993-05-27 2005-09-15 Board Of Regents Of The Univ Of Washington サイクリックｇｍｐ結合性、サイクリックｇｍｐ特異的ホスホジエステラーゼの材料および方法
US20070275428A1 (en) * 2006-05-23 2007-11-29 Gambhir Sanjiv S Protein phosphorylation imaging systems, methods of making phosphorylation imaging systems, and methods of use thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005245467A (ja) * 1993-05-27 2005-09-15 Board Of Regents Of The Univ Of Washington サイクリックｇｍｐ結合性、サイクリックｇｍｐ特異的ホスホジエステラーゼの材料および方法
WO2000071668A2 (fr) * 1999-05-20 2000-11-30 The Board Of Regents Of The University Of Nebraska Procede d'identification de composants impliques dans les mecanismes de transduction de signaux dans les plantes superieures
JP2003509051A (ja) * 1999-09-15 2003-03-11 プロメガ コーポレイション フォツリス・ペンシルバニカ及びピロフィルス・プラギオフタラムス由来の熱安定性ルシフェラーゼ及び製造方法
US20070275428A1 (en) * 2006-05-23 2007-11-29 Gambhir Sanjiv S Protein phosphorylation imaging systems, methods of making phosphorylation imaging systems, and methods of use thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Dai 3 Kai Riken·Bunshiken Godo Symposium Extreme Photonics Research -Extreme Hacho no Hassei to Oyo-, 2006", article MASAKI TAKEUCHI: "Luciferase no Kozo Joho ni Motozuku Kinosei Hakko Bunshi no Kaihatsu", pages: 129 *
"Dai 5 Kai Riken·Bunshiken Godo Symposium Extreme Photonics Research -Bioimaging-, 2007", article MASAKI TAKEUCHI: "Luciferase Saikosei o Mochiita cGMP Kenshutsu Hakko Probe no Kaihatsu", pages: 65, 66 *
MASAKI TAKEUCHI: "Seisaibonai cGMP o Hakko Kenshutsu suru Probe Bunshi no Kaihatsu", JOURNAL OF JAPANESE BIOCHEMICAL SOCIETY, 4 December 2008 (2008-12-04), pages 3T26 - 5 *
YASUTAKA NAGAOKA: "Seisaibonai no Cyclic GMP o Kashika suru Hakko Probe no Kaihatsu", THE CHEMICAL SOCIETY OF JAPAN KOEN YOKOSHU, vol. 89TH, no. 2, 13 March 2009 (2009-03-13), pages 1294 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2436764A4 (fr) * 2009-05-29 2012-11-28 Univ Tokyo Procédé de détection ultrasensible d'une interaction protéine-protéine
JP2011195547A (ja) * 2010-03-23 2011-10-06 Olympus Corp サイクリックｇｍｐ解析方法

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