JP2016161420A - Juvenile hormone sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop and provide a juvenile hormone sensor capable of detecting and quantifying juvenile hormones at high sensitivity.SOLUTION: A juvenile hormone-specific biosensor protein is provided, which is a juvenile hormone-binding protein of lepidopteran origin modified or linked, at a specific side, with two types of phosphors having different fluorescence characteristics, or the like. When a juvenile hormone binds with the sensor protein, a conformational change of the juvenile hormone-binding protein causes a resonance energy transfer (FRET) between the phosphors. High-sensitivity detection and quantification of the juvenile hormone can be done by detecting the change in energy.SELECTED DRAWING: None

Description

  The present invention relates to a juvenile hormone sensor capable of detecting and quantifying juvenile hormone in and outside the living body, a method for monitoring in vivo kinetics of juvenile hormone, and a method for searching juvenile hormone analogs using the juvenile hormone sensor About.

  Juvenile Hormone (JH) (hereinafter often referred to as “JH”) is a hormone unique to insects and other arthropods, and is a physiological phenomenon in all stages from eggs to adults. Is involved in. After expression, JH is secreted into the blood via intracellular transport such as signal peptide removal. After that, it moves in the blood and is carried to the target cell or tissue, and binds to the JH receptor to exert its function (Non-Patent Document 1).

  JH analogs (JH-like compounds) that mimic the molecular structure of JH can effectively inhibit metamorphosis of arthropods such as insects. On the other hand, it has no effect on mammals including humans or is extremely low, so it is used as an active ingredient such as agricultural chemicals as a highly safe insecticide. However, since development and search for JH analogs with high pharmacological effects involve technical difficulties, new JH analogs have not been obtained in recent years. In general, in order to effectively and efficiently develop new JH analogs, a high-throughput ligand screening method that can detect and quantify JH simply, rapidly, and with high sensitivity is required. However, a method for screening JH with high throughput has not been established so far.

  Conventionally, high-performance liquid chromatograph-mass spectrometry (LC-MS) method (Non-Patent Documents 2 and 3), radioisotope (RI) method (Non-Patent Document 4), or electrochemical impedance spectroscopy is used for detection and quantification of JH. The (EIS) method (Non-Patent Document 5) has been used. However, the detection and quantification of JH by the LC-MS method has a problem that complicated JH extraction processing is required and JH needs to be converted into a derivative for measurement with high sensitivity. In addition, the JH quantification by the RI method has a problem that an RI facility is required and it takes time to obtain a result. Furthermore, JH quantification by the EIS method has only one reported example, and there are problems in terms of reproducibility and versatility. In addition to these, as a new detection and quantification method of JH, a method using “screening system for novel insect control agent using JH response element” (Non-patent Document 6) is known. This method is technically more useful than the conventional method, but has problems of cost and indirect JH determination.

  Therefore, none of the conventional JH detection and quantification methods are suitable as a high-throughput screening method, and the development of a simpler, faster and more sensitive JH detection and quantification method has been demanded.

  Furthermore, in order to verify the pharmacological effects of JH analogs in vivo, a method for monitoring the pharmacokinetics of JH is indispensable, but no such method has been known to date. For example, each of the methods for detecting and quantifying the above-mentioned JH involves a cell or tissue destruction operation such as extraction of body fluids or tissues, and thus there is a problem that it is not possible to obtain information on the dynamics of JH in vivo. .

Manabu Kamimura, Tetsuro Shinoda, 2008, Protein Nucleic Acid Enzyme 53: 105-110 Westerlund S.A. and Hoffmann K.H., 2014, Anal. Bioanal. Chem., 379: 540-543 Furuta K., et al., 2013, Biosci. Biotechnol. Biochem., 77 (5): 988-991 Vermunt A.M.W., et al., 2001, Insect Mol. Biol. 10: 147-154 Stobiecka A., et al., 2008, Front. Biosci. 13: 2866-2874 Kayukawa T., et al., 2012, Proc Natl Acad Sci USA, 109 (29): 11729-11734

  An object of the present invention is to develop and provide a JH biosensor capable of detecting and quantifying JH with high sensitivity in vitro and in vivo, and using the JH sensor to produce a highly effective JH analog at high throughput. It is intended to provide a screening method and a method for monitoring the in vivo kinetics of JH.

  JH acts on the whole body through body fluids, but cannot move through body fluids alone because of its poor water solubility. Therefore, in lepidopterous insects, JH is stored inside a juvenile hormone binding protein (JHBP) (hereinafter referred to as “JHBP” in this specification), and a complex state (JHBP-JH complex) ) Makes it possible to move in body fluids.

  The present inventors previously performed a three-dimensional structure analysis of JHBP and JHBP-JH complex, and revealed that the three-dimensional structure of JHBP is changed by the binding of JH (Suzuki et al. 2011, Sci. Rep. 1: 133. DOI: 10.1038 / srep00133). In order to solve the above problems, the present inventors have focused on the three-dimensional structure change of JHBP due to the binding of JH, and as a result of repeated research, as a result of research, two kinds of fluorescent substances having different fluorescence characteristics were added to specific positions of JHBP. It was found that only when incorporated could JH binding be detected by fluorescence resonance energy transfer (FRET). Based on this result, we have developed a JH-specific biosensor protein that can detect and quantify JH with high sensitivity and analyze the dynamics of JH in vivo. The present invention is based on the research and development results, and provides the following.

(1) In the truncated amino acid sequence excluding the signal peptide of JHBP in Lepidoptera, when the amino acid residue at the amino terminus of the truncated amino acid sequence is defined as position 1, any one amino acid residue at positions 10-15 Is modified by any one of the resonance energy donating substance and the resonance energy accepting substance that receives the excitation energy of the resonance energy donating substance, and the amino acid residue at the carboxyl terminal of the truncated amino acid sequence is the resonance energy donating substance A JH sensor comprising a substance and a protein modified with the other of the resonance energy accepting substance.
(2) The JH sensor according to (1), wherein the resonance energy donating substance is a luminescence or fluorescence resonance energy donating substance.
(3) In the truncated amino acid sequence excluding the signal peptide of JHBP in Lepidoptera insects, when the amino acid residue at the amino terminus of the truncated amino acid sequence is defined as position 1, two consecutive amino acids within the range of positions 10-15 Any one of a resonance energy donating substance and a resonance energy accepting substance that accepts excitation energy of the resonance energy donating substance is linked between amino acid residues, and the resonance energy donating type is connected to the carboxyl terminus of the truncated amino acid sequence. A JH sensor comprising a fusion polypeptide in which the other of the substance and the resonance energy accepting substance is linked.
(4) The JH sensor according to (3), wherein the resonance energy donating substance and / or the resonance energy accepting substance is a peptide.
(5) The JH sensor according to (4), wherein the peptide is a luminescent or fluorescent peptide.
(6) In the truncated amino acid sequence excluding the signal peptide of juvenile hormone binding protein in Lepidoptera insects, when the amino acid residue at the amino terminus of the truncated amino acid sequence is defined as position 1, it is within the range of positions 10-15. The amino-terminal amino acid sequence of the luminescent or fluorescent protein fragment divided in two between two consecutive amino acid residues is linked, and the carboxy-terminal of the luminescent or fluorescent protein fragment divided into two at the carboxyl terminus of the truncated amino acid sequence JH sensor consisting of a fusion polypeptide with linked amino acid sequences.
(7) The JH sensor according to any one of (3) to (6), wherein the distance between the two consecutive amino acid residues is between the 11th and 12th amino acid residues.
(8) The JH sensor according to any one of (3) to (7), wherein the truncated amino acid sequence is any amino acid sequence represented by (a) to (c).
(A) an amino acid sequence represented by SEQ ID NO: 2, 3, 6 or 8,
(B) an amino acid sequence in which one or more amino acid residues are deleted, substituted or added in any one of the amino acid sequences described in (a), or (c) any one of the amino acid sequences described in (a) DNA encoding the fusion polypeptide constituting the JH sensor according to any one of amino acid sequences (9), (4) to (8) having 90% or more amino acid identity to the amino acid sequence.
(10) A JH sensor expression vector comprising the DNA of (9) in an expressible state.
(11) A transformant obtained by introducing the JH sensor expression vector according to (10) into a host cell or host organism, or a progeny thereof.
(12) A method for monitoring the dynamics of JH in an arthropod in vivo, the step of introducing the JH sensor according to any one of (1) to (8) into the arthropod of the subject, the test A step of measuring luminescence or fluorescence generated by reorganization of luminescence or fluorescent protein, or an energy variation that fluctuates due to resonance energy transfer in the body, and a step of detecting JH based on the measurement value measured in the measurement step Method.
(13) The method according to (12), wherein the fluctuating energy mutation is luminescence or fluorescence.
(14) A method for monitoring the dynamics of JH in an arthropod in vivo, the step of introducing a JH sensor expression vector according to (10) into a subject arthropod to produce a transformant, In the transformant, a step of measuring luminescence or fluorescence generated by reorganization of an energy mutation or fluorescence protein derived from a luminescence or fluorescence resonance energy transfer derived from a JH sensor expressed from a JH sensor expression vector in the transformant, and the measurement step Detecting the JH based on the measured values.
(15) The method according to (14), wherein the fluctuating energy mutation is luminescence or fluorescence.
(16) A method for searching for JH analogs structurally similar to arthropod JH, the step of mixing the JH sensor according to any one of (1) to (8) and a candidate substance, JH sensor A step of measuring fluorescence generated by energy variation due to resonance energy transfer of luminescence or reconstruction of luminescent or fluorescent protein, and a step of selecting a candidate substance as a JH analog based on the measurement value obtained in the measurement step Method.
(17) The method according to (16), wherein the fluctuating energy mutation is luminescence or fluorescence.
(18) In (16) or (17), wherein the selection step is based on a significant difference from a measurement value of a negative control in which no candidate substance is mixed in the mixing step, or an increase in the concentration-dependent measurement value of the candidate substance The method described.

  According to the JH sensor of the present invention, JH can be detected and quantified with high sensitivity in vitro and in vivo.

  According to the JH analog search method of the present invention, it is possible to provide a system for screening a JH analog with high throughput using the JH sensor.

  According to the in vivo kinetic monitoring method of JH of the present invention, the kinetics of JH in a living body such as an individual, a tissue, or a cell can be monitored without performing a cell or tissue destruction operation.

It is a figure which shows the change by the three-dimensional structure of JHBP and the coupling | bonding of JH. A: The three-dimensional structure of JHBP when JH is not bound. When JH is not bound, the molecular door of JHBP is opened and the JH binding pocket in the molecule is open. B: A three-dimensional structure of a JHBP-JH complex in which JH is bound to JHBP. The molecular door is closed by JH binding to the JH binding pocket, and JH is stored in the JHBP molecule. It is the figure which aligned the amino acid sequence of JHBP in the Lepidoptera insect. In the figure, BmJHBP stands for Bombyx mori JHBP, MsJHBP stands for Manduca sexta JHBP, HvJHBP stands for Heliothis virescens JHBP, and GmJHBP stands for Galleria mellonella JHBP. These JHBPs are all amino acid sequences of mature JHBP from which the amino terminal signal peptide has been cleaved. In the figure, the number at the top of the nuclear sequence indicates the position of each amino acid residue when the amino acid residue on the amino terminal side of mature JHBP is defined as position 1. In addition, when 4 types of JHBP are aligned, residues in which amino acid identity is found in 3 or more of the 4 types are shaded. Furthermore, the amino acid sequences of various JHBPs surrounded by a frame are the range of amino acid residues modified by the resonance energy donating substance or the resonance energy accepting substance, the resonance energy donating substance or the resonance energy accepting substance in the JH sensor of the present application. Is a range of amino acid residues to which is linked, or a range to which amino-terminal fragments of luminescent or fluorescent proteins divided in two are linked. A cloning vector for constructing a JH sensor is shown. A is a plasmid map of mTFPmVenus_pRSET-A (Kpn) _ver1.2, and B is a plasmid map of mTFPmVenus_pRSET-A (RV) _ver1.2. The schematic diagram of each FRET-JHBP construct constructed in Example 1 is shown. FRET-JHBP-TypeA is a construct having the configuration of the JH sensor of the present invention. It is a figure which shows the presence or absence of FRET in the presence and absence of JH by each FRET-JHBP construct constructed in Example 1. A is the result of using FRET-JHBP-TypeA, B is FRET-JHBP-TypeB, and C is FRET-JHBP-TypeC as a FRET-JHBP construct. It is a figure which shows a time-dependent change of FRET in the presence of various JH or JH analog when FRET-JHBP-TypeAd5 is used as a JH sensor. In the figure, “met. Acid” represents metoprenic acid, and “MeFa” represents methyl farnesate (MF). It is a figure which shows the relationship between the density | concentration of various JH or a JH analog when FRET-JHBP-TypeAd5 is used as a JH sensor, and FRET.

Each embodiment of the present invention will be specifically described below.
1. Juvenile hormone sensor 1-1. Outline | summary The 1st aspect of this invention is a juvenile hormone sensor (JH sensor). The JH sensor of this embodiment is a biosensor that utilizes the three-dimensional structure change of juvenile hormone-binding protein (JHBP) derived from Lepidoptera by binding of juvenile hormone, based on the principle of resonance energy transfer of FRET, or It is characterized by sensitive detection and quantification of arthropod JH in vitro and in vivo based on the reconstruction of fluorescent or luminescent proteins.

1-2. Constitution The JH sensor of the present invention is (1) a modification of a resonance energy donating substance and a resonance energy accepting substance capable of causing resonance energy transfer to amino acid residues at two different specific positions of JHBP derived from Lepidoptera insects. Or (2) a fusion polypeptide obtained by linking a resonance energy donating substance and a resonance energy accepting substance to two different positions of JHBP derived from Lepidoptera, or (3) Lepidoptera It consists of a fusion polypeptide formed by linking an amino-terminal fragment and a carboxyl-terminal fragment of a luminescent or fluorescent protein fragment divided into two at two different specific positions of insect-derived JHBP.

  In the present specification, the “arthropod” is a species belonging to the taxonomic arthropoda (Arthropoda). An arthropod is composed of a plurality of somites covered with an exoskeleton made of chitin, and is characterized by growing by molting. Arthropods include, for example, organisms (insects) belonging to the insect class (Insecta), organisms (crustacean organisms) belonging to the crustacea (Crustacea), organisms belonging to the arachnid class (Arachnida), etc. included. Insects include Lepidoptera organisms, Coleoptera organisms, Diptera organisms, Hymenoptera organisms, Hemiptera organisms, Orthoptera organisms, and the like. Crustaceans include shrimp (Decapoda) organisms and the like. The arachnid organisms include Acari organisms, Araneae organisms, and Scorpionida organisms.

  In the present specification, “juvenile hormone (JH): Juvenile Hormone” is a substance that is the main target of detection and quantification of the JH sensor of the present invention. JH is a compound having a sesquiterpenoid skeleton, and functions as a hormone involved in all stages of physiological phenomena from egg to adult, such as control of embryonic development, molting / metamorphosis, reproduction, dormancy, and longevity of arthropods . In general, JH in insects is well known, but JH in this specification is not only a narrow definition of JH in insects, but also a concept that indicates JH in a broad sense including sesquiterpenoid derivatives involved in the above physiological phenomena in arthropods It is. Therefore, methyl farnesoate (MF) and the like in crustaceans are also included in JH in this specification. In addition, JH I, 4-methyl JH I, JH II, JH III, JHB3, and JHSB3 are known as JH in insects, and since any JH can bind to JHBP described later, Can be a target for JH sensors.

  In insects, JH is biosynthesized in glandular tissue called corpora-allata (CA) at the rear end of the head and secreted into the blood. As described above, since JH is a compound that is hardly soluble in water, a transport protein is required for JH to reach target cells via blood.

  In this specification, “juvenile hormone binding protein (JHBP): Juvenile Hormone Binding Protein” is a transport protein present in Lepidoptera that binds to JH secreted in the blood and transports JH to the target organ. is there. As shown in FIG. 1, JHBP has a JH binding pocket in the molecule. Before JH binding, the molecular door of this JH binding pocket is open (Fig. 1A), and the molecular door is closed by JH binding and JH is stored in the molecule (Fig. 1B). (Suzuki et al. 2011, supra). JHBP controls the normal development and growth of Lepidoptera insects by regulating the protection of JH from degrading enzymes, blood transport, and storage.

  JHBP is expressed as a JHBP precursor consisting of a total length of 230 to 260 amino acids having a signal peptide (signal sequence) on the amino terminal side. Thereafter, through intracellular transport, the signal peptide is cleaved and removed to form a mature JHBP (secreted JHBP) consisting of a truncated amino acid sequence and secreted outside the cell. Specific examples of the JHBP precursor include a silkworm (Bombyx mori) JHBP precursor consisting of the amino acid sequence represented by SEQ ID NO: 1. In the silkworm JHBP precursor consisting of 243 amino acids in total length, 18 amino acid residues from the amino terminus including the initiation methionine correspond to the signal peptide. Accordingly, the mature JHBP of silkworm consists of the amino acid sequence shown in SEQ ID NO: 2 (225 amino acids) excluding the signal peptide. In this specification, unless otherwise specified, when JHBP is indicated, it means “mature JHBP” from which the signal peptide has been removed, and JHBP containing the signal peptide is referred to as “JHBP precursor”.

  In this specification, “resonance energy donating substance” (herein often referred to as “donor type substance”) and “resonance energy accepting type substance” (herein often referred to as “acceptor type substance”). ) Means a substance consisting of two types and one set that can cause resonance energy transfer between both substances.

  "Resonance energy transfer" is a FRET (Forster resonance energy) in which excitation energy such as fluorescence or luminescence generated in a donor-type material is transferred to the acceptor-type material when the donor-type material and the acceptor-type material are located in the physical vicinity. Transfer or Fluorescence resonance energy transfer). In FRET, the emission or fluorescence spectrum of the donor-type substance and the absorption spectrum of the acceptor-type substance partially overlap. In this specification, FRET is not only a radiation reaction in which an acceptor-type substance generates a sensitization reaction by the excitation energy of a donor-type substance, and the acceptor-type substance emits fluorescence or light emission, but the excitation energy of the donor-type substance is an acceptor type. It also includes a non-radiative reaction (so-called quenching reaction) in which the acceptor type substance does not emit fluorescence or luminescence, although it moves to the substance. In the latter case, fluorescence and light emission from the donor-type substance disappear due to the transfer of excitation energy, and as a result, the acceptor-type substance functions as a quencher for the donor-type substance.

  As described above, the donor-type substance and the acceptor-type substance are substances that can cause the FRET phenomenon in principle. However, in the present specification, the substance itself is not a substance that can cause the FRET phenomenon, but includes a substance that can carry a substance that can cause the FRET phenomenon. As used herein, “supporting” refers to addition or modification. Examples of the substance capable of supporting a substance capable of causing the FRET phenomenon include amino acids (including natural amino acids and non-natural amino acids). For example, in the amino acid sequence of JHBP, an amino acid irrelevant to the amino acid sequence of JHBP is linked between the amino acid residues at the specific positions described later and the C-terminal amino acid residue, and during or after the synthesis of the JH sensor of the present invention, By supporting a substance capable of causing the FRET phenomenon on the amino acid, that is, a fluorescent dye molecule, a luminescent molecule, or the like, the amino acid can eventually become a donor-type substance and an acceptor-type substance.

  The donor substance and the acceptor substance are not particularly limited as long as they can cause the FRET phenomenon. Examples thereof include fluorescent dye molecules or luminescent molecules, and resonance energy donating or accepting peptides.

  The fluorescent dye molecule or the light emitting molecule as the donor-type substance and the acceptor-type substance is not particularly limited as long as it has the above properties. The efficiency of FRET depends on the size, distance, and angle of the overlap of the emission or fluorescence spectrum of the donor-type substance and the acceptor-type substance, so that the excitation spectrum of the known fluorescent dye molecule or luminescent molecule, and the emission or fluorescence Based on the spectrum, it may be determined as appropriate while taking into account the overlap of the spectra of the two substances. For example, a combination using FITC or FAM as a donor-type substance and TRITC, Rhodamin or TAMURA as an acceptor-type substance, or a combination using Cy3, Rhodamine or TAMURA as a donor-type substance and Cy5 as an acceptor-type substance can be mentioned. In addition, there is a combination in which EDANS or 6-FAM is used as a donor-type substance and a quencher DABCYL is used as an acceptor-type substance.

  In the present specification, “resonance energy donating peptide” (herein often referred to as “donor type peptide”) and “resonance energy accepting peptide” (herein often referred to as “acceptor type peptide”). ) Refers to a peptide consisting of a set of two species capable of causing resonance energy transfer between peptides, such as a protein. The principle of resonance energy transfer is as described above. This is a FRET phenomenon in which excitation energy such as fluorescence or luminescence generated by a donor-type peptide is transferred to an acceptor-type peptide, and particularly includes bioluminescence resonance energy transfer (BRET).

  The donor-type peptide and the acceptor-type peptide may be appropriately determined in consideration of the spectrum overlap of both peptides, as in the case of the donor-type substance and the acceptor-type substance, and are not particularly limited. For example, donor protein / acceptor protein pairs include mTFP1 / mVenus, CFP / YFP, GFP / mCherry, Clover GFP / mRuby, and luciferase / GFP. The amino acid sequence information of these proteins is well known in the art, and searches such as the Protein search site (http://www.ncbi.nlm.nih.gov/protein) on the NCBI (National Center for Biotechnology Information) homepage It can be easily obtained by using the site. For example, in the case of the amino acid sequence of mTFP1, the amino acid sequence represented by SEQ ID NO: 10 is exemplified, and in the case of mVenus, the amino acid sequence represented by SEQ ID NO: 12 is exemplified.

  Further, the combination is not limited to peptides, and may be a combination of a peptide and a fluorescent dye molecule or a luminescent molecule, a combination of a peptide and a quencher, or the like.

  “Reconstruction of fluorescent or photoprotein” means that when each of the two divided luminescent or fluorescent protein fragments is physically close to each other, it is reconstructed by self-assembly, and luminescent or fluorescent radioactivity is increased by structural complementation. It means to recover.

  In this specification, “a luminescent or fluorescent protein fragment that has been divided into two parts” (in this specification, often referred to as “two-divided protein fragment”) refers to one luminescent protein or fluorescent protein in the amino-terminal region. A polypeptide fragment divided into two regions (often referred to as “N-terminal fragment” in this specification) and a carboxyl-terminal region (often referred to as “C-terminal fragment” in this specification). is there. The photoprotein and the fluorescent protein are not particularly limited. For example, GFP or the like can be used for a fluorescent protein, and luciferase or the like can be used for a luminescent protein. Specific examples include split GFP and split Renilla luciferase (Ozawa T. et al., 2001, Anal Chem., 73 (11): 2516-21) (Ozawa T. et al., 2006, Anal Chim Acta., 556 (1): 58-68). Moreover, the split fluorescent protein system described in Japanese translations of PCT publication No. 2007-508841 can also be utilized. Regarding the position on the amino acid sequence that divides the luminescent or fluorescent protein in two, it is the position where luminescence or fluorescence is lost by the two divisions, and the structure by physical proximity of the N-terminal fragment and C-terminal fragment generated by the two division The position is not particularly limited as long as it is a position where light emission or fluorescence can be recovered by the complementary operation.

  The JH sensor of this embodiment is based on mature JHBP consisting of a truncated amino acid sequence obtained by removing a signal peptide from the full-length amino acid of a JHBP precursor. Thus, the amino acid residue located at the amino terminus of JHBP is not the initiating methionine. Specific examples of such JHBP include silkworm JHBP consisting of the amino acid sequence shown in SEQ ID NO: 2 above, and other species JHBP belonging to the same Lepidoptera insect, for example, JHBP of tobacco tin mega (Manduca sexta) shown in SEQ ID NO: 4. , JHBP of Heliothis virescens represented by SEQ ID NO: 6, and JHBP of Galleria mellonella represented by SEQ ID NO: 8, but are not limited thereto.

  In addition, JHBP is a mutant JHBP consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted or added in the amino acid sequences of various wild-type JHBPs in Lepidoptera insects, or various types of Lepidoptera insects. Variant JHBP consisting of an amino acid sequence having 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity to the amino acid sequence of wild-type JHBP It may be. In the present specification, the term “plurality” means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. “Amino acid identity” refers to the alignment of two amino acid sequences (alignment) and introducing a gap in one of the amino acid sequences as necessary so that the amino acid identity between the two is the highest. The ratio (%) of the same amino acid residue to the number of amino acid residues of wild-type JHBP. Amino acid identity can be calculated using a protein search system by BLAST or FASTA (Karlin, S. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 5873-5877; Altschul, S. F. et al., 1990, J. Mol. Biol., 215: 403-410; Pearson, WR et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448 ). The mutant JHBP has substantially the same properties as wild-type JHBP. Therefore, it is desirable that the mutated amino acid residue and mutation position in the amino acid sequence of the mutant JHBP have properties or positions that do not affect the three-dimensional structure of JHBP and JH binding ability. For example, in the case of the amino acid substitution, substitution within a conservative amino acid having similar properties such as charge, side chain, polarity and aromaticity is desirable. Specifically, in basic amino acids (arginine, lysine, histidine), in acidic amino acids (aspartic acid, glutamic acid), in non-polar polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), nonpolar Amino acids within amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched chain amino acids (leucine, valine, isoleucine), or aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine) Substitution may be mentioned. Moreover, if it is the said amino acid deletion or addition, it will be set as the position which does not destroy the three-dimensional structure and JH binding ability of wild type JHBP. For example, in SEQ ID NO: 2, positions 173 to 186 correspond. This position corresponds to the lower region of JHBP opposite to the upper region of JHBP in which the JH binding site forming the JH binding pocket is present in the three-dimensional structure of JHBP shown in FIG.

  As shown in FIG. 2, JHBP constituting the JH sensor of the present invention has a relatively low conservation of amino acid homology between Lepidoptera insect species of about 40 to 50%. However, the three-dimensional structure and amino acid residues that recognize JH are close to each other. Therefore, the identification of heterologous orthologues of JHBP is not based solely on interspecies conservation of amino acid sequences with known JHBPs described below, but the three-dimensional structure predicted from the amino acid sequences is similar to the three-dimensional structure of known JHBPs. Judgment is made in consideration of points.

  In this specification, “specific position of JHBP” means (1) the position of a specific amino acid residue on the amino acid sequence of JHBP that modifies the donor-type substance and acceptor-type substance, and (2) the donor-type substance and acceptor-type. It refers to a specific position on the amino acid sequence of JHBP that links the substances, or (3) a specific position on the amino acid sequence of JHBP that links the N-terminal fragment and the C-terminal fragment. In (1), the amino acid sequence of JHBP is not altered, but in the cases of (2) and (3), the amino acid sequence of JHBP is altered. In order to detect the three-dimensional structural change of JHBP due to JH binding based on the FRET principle or the reconstruction of fluorescent proteins, etc., JH binding to JHBP is not inhibited, and FH is generated by the three-dimensional structural change of JHBP. The donor-type and acceptor-type substances must be modified or linked to positions, or positions that cause structural complementation, or N-terminal and C-terminal fragments must be linked. Therefore, the specific position of the JHBP is the most important feature in the JH sensor of the present invention. Hereinafter, each of the cases (1) to (3) will be specifically described.

(1) When a specific amino acid residue on the amino acid sequence of JHBP is modified with a donor-type substance and an acceptor-type substance When the amino terminal amino acid residue of mature JHBP is defined as position 1, any of positions 10-15 This single amino acid residue and the amino acid residue at the carboxyl terminal correspond to a specific position of JHBP.

  In the present specification, among the “any one amino acid residue within the range of positions 10 to 15”, the specific position of JHBP may be any position as long as it is a modifiable amino acid residue. Here, “modifiable amino acid residues” include, for example, Cys residues, Lys residues, Tyr residues, Trp residues, His residues and the like. In addition, a non-natural amino acid designed to be easily modified by the Click reaction may be linked to a specific position of JHBP as a donor-type substance or an acceptor-type substance. The method for modifying the donor-type substance or the acceptor-type substance with a predetermined amino acid residue is not particularly limited, and a known method may be used. Examples thereof include a bioconjugation method, a bioorthogonal reaction method, and a post-affinity labeling modification (P-ALM) / affinity-based labeling method.

  When the JH sensor of the present invention is modified with a donor-type substance and an acceptor-type substance, the modification order of each substance is not limited. For example, the donor-type substance may be modified with any one amino acid residue within the range of positions 10 to 15 of JHBP, or the amino acid residue at the carboxyl terminal of JHBP may be modified. Once the modification position of the donor-type substance is determined, the remaining amino acid residue at the other position is necessarily modified with the acceptor-type substance.

(2) When a donor-type substance and an acceptor-type substance are linked to a specific position on the amino acid sequence of JHBP When the amino terminal amino acid residue of mature JHBP is defined as position 1, it is within the range of positions 10-15. Between two consecutive amino acid residues and the carboxyl terminus of JHBP correspond to a specific position of JHBP.

  In the present specification, “between two consecutive amino acid residues within the range of positions 10 to 15” specifically refers to the position between positions 10 and 11 (referred to as “position 10/11”; the same applies hereinafter). , 11th / 12th place, 12th / 13th place, 13th / 14th place, and 14th / 15th place. Preferably between 11th and 12th. For example, in the case of silkworm JHBP consisting of the amino acid sequence shown in SEQ ID NO: 2, lysine at position 10 / leucine at position 11 (denoted as “K10 / L11”; hereinafter the same), between L11 / G12, G12 / Between D13, between D13 / M14, and between M14 / Q15, preferably between L11 / G12.

  There is no limitation on the order of connection between the donor type material and the acceptor type material. For example, the donor-type substance may be linked between two consecutive amino acid residues within the range of positions 10 to 15 of JHBP, or may be linked to the carboxyl terminus of JHBP. If the connection position of the donor material is determined, the acceptor material is connected to the remaining position.

(3) When the JH sensor contains an N-terminal fragment and a C-terminal fragment of a bipartite protein fragment, as in (2), when the amino terminal amino acid residue of mature JHBP is defined as position 1, Between two consecutive amino acid residues within the range of position 15 and the carboxyl terminus of JHBP correspond to a specific position of JHBP.

  Unlike (1) and (2) above, the linking order of the N-terminal fragment and the C-terminal fragment is fixed, and the N-terminal fragment is a residue of two consecutive amino acids within the range of positions 10-15 of JHBP. Linked between groups, the C-terminal fragment must be linked to the carboxyl terminus of JHBP.

1-3. Effect Since the JH sensor of the present invention has high specificity for JH, JH can be detected and quantified with high sensitivity. In particular, MF, which is JH of the crustacean organism, is difficult to ionize because it does not have an epoxy ring, and quantification by mass spectrometry was extremely difficult, but according to the JH sensor of the present invention, highly sensitive detection and quantification of trace amounts of MF Is also possible.

The JH sensor of the present invention can detect in vivo JH at a 10 ⁻⁸ concentration level without destroying cells or tissues.

  The JH sensor of the present invention can enable high-throughput ligand screening in the development of novel JH analogs and the like.

  The JH sensor of the present invention can be applied not only to any species of arthropods that express JH, but also to microorganisms, plants, vertebrates and the like that do not have JH. This makes it possible to study the mechanism of JH translocation and metabolism in vivo.

2. DNA encoding juvenile hormone sensor
2-1. Overview The second aspect of the present invention is DNA encoding the JH sensor of the first aspect (often referred to herein as “JH sensor DNA”). The JH sensor DNA of this embodiment corresponds to the JH sensor of the first embodiment, in which the sensor is a JHBP fusion polypeptide containing a donor peptide and an acceptor peptide.

2-2. Constitution
In the JH sensor DNA, the base sequence of DNA encoding JHBP (often referred to as “JHBP gene” in this specification) includes a JHBP gene encoding silkworm JHBP consisting of the amino acid sequence shown in SEQ ID NO: 2, specifically For example, the silkworm JHBP gene consisting of the base sequence shown in SEQ ID NO: 3, the JHBP gene encoding tobacco tin mega JHBP consisting of the amino acid sequence shown in SEQ ID NO: 4, specifically, for example, the base sequence shown in SEQ ID NO: 5 Tobacco tin mega JHBP gene consisting of: JHBP gene encoding the tobacco tobacco JHBP consisting of the amino acid sequence shown in SEQ ID NO: 6, specifically, eg, the tobacco tobacco JHBP gene consisting of the nucleotide sequence shown in SEQ ID NO: 7, and shown in SEQ ID NO: 8 JHBP gene coding for Hachinotsu Riga JHBP consisting of an amino acid sequence, specifically, for example, the nucleotide sequence represented by SEQ ID NO: 9 Ranaru Hachinosutsudzuriga JHBP gene, and the like. The JHBP gene may be a mutant JHBP gene encoding the mutant JHBP of each species described in the first embodiment, in addition to the wild-type JHBP gene.

  In JH sensor DNA, DNA encoding donor peptide and acceptor peptide, or DNA encoding N-terminal fragment and C-terminal fragment of bipartite protein fragment is derived from the base sequence encoding each peptide or protein fragment. Any DNA can be used. The nucleotide sequence information of DNA encoding these peptides is also known in the art. For example, it can be easily obtained by using a search site such as the Gene search site (http://www.ncbi.nlm.nih.gov/gene) on the NCBI homepage. Specifically, the base acid sequence represented by SEQ ID NO: 11 as the base sequence of DNA encoding mTFP1 comprising the amino acid sequence represented by SEQ ID NO: 10 also encodes mVenus comprising the amino acid sequence represented by SEQ ID NO: 12. An example of the base sequence of DNA is the base acid sequence represented by SEQ ID NO: 13.

  The JH sensor DNA of the present invention may be ligated with DNA encoding a signal peptide (often referred to as “signal DNA” herein) at the 5 ′ end. A signal peptide is a peptide necessary for extracellular transfer of a protein biosynthesized in a cell. By fusing the signal peptide to the amino terminus of the JH sensor, the JH sensor can be expressed as a secreted protein.

  The amino acid sequence of the signal peptide is not particularly limited. Any signal peptide that can function in the arthropod in vivo can be utilized. The amino acid length of the signal peptide is not particularly limited. It may be within the range of 3 to 60 amino acids, preferably 7 to 30 amino acids, 8 to 25 amino acids, or 9 to 20 amino acids. Therefore, the signal DNA may be in the range of 9 to 120 bases, preferably 21 to 90 bases, 24 to 75 bases, or 27 to 60 bases. In general, a signal peptide having a short amino acid length is preferred. A specific example of the signal peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 14. This signal peptide corresponds to a peptide consisting of 18 amino acid residues from the amino terminus including the starting methionine of the silkworm JHBP precursor consisting of the amino acid sequence shown in SEQ ID NO: 2. Examples of the DNA encoding the signal peptide of the silkworm JHBP precursor include a DNA comprising the base sequence represented by SEQ ID NO: 15.

2-3. Preparation of JH sensor DNA The JH sensor DNA of the present invention encodes JHBP and a luminescent or fluorescent protein as a donor peptide and an acceptor peptide, or an N-terminal fragment and a C-terminal fragment of a bipartite protein fragment, respectively. DNA can be prepared by ligation based on molecular genetic techniques known in the art.

  JHBP-encoding DNA is, for example, based on the nucleotide sequence information shown in SEQ ID NO: 2, 4, 6 or 8, or the nucleotide sequence information obtained from a search site such as a Gene search site on the NCBI website. Using a partial sequence or a partial sequence complementary thereto as a probe or primer, it can be obtained from a cDNA library of an appropriate species by PCR or the like.

  The signal DNA can be chemically synthesized based on the known signal DNA base sequence information, or can be obtained from a gene containing the signal DNA by PCR or the like.

  The DNA encoding each of the luminescent or fluorescent protein donor-type protein and acceptor-type protein or the N-terminal fragment and C-terminal fragment of the bipartite protein fragment can be prepared in the same manner as the DNA encoding JHBP. For example, based on the base sequence information obtained from a search site such as the Gene search site described above, using a partial sequence of the base sequence or a partial sequence complementary thereto as a probe or primer, from a plasmid or the like containing the DNA It may be prepared by PCR or the like. Plasmids containing these DNAs are commercially available and can be used.

  JH sensor DNA is prepared by first encoding the JHBP-encoding DNA between codons encoding two consecutive amino acids within the range of positions 10 to 15 of JHBP, specifically, the base at the 5 ′ end of the JHBP-encoding DNA. Is the 1st position, between the 30th and 31st bases, between the 33rd and 34th bases, between the 36th and 37th bases, between the 39th and 40th bases, and between the 42nd and 43rd positions Between these bases, DNA encoding either the donor protein or the acceptor protein or the DNA encoding the N-terminal fragment of the bipartite protein fragment is ligated so that the reading frame matches. Next, the DNA encoding the other of the donor-type protein and the acceptor-type protein or the DNA encoding the C-terminal fragment of the bipartite protein fragment is ligated to the 3 'end of the DNA encoding JHBP so that the reading frame matches. When ligating the signal DNA, it may be ligated so that the reading frame matches the 5 'end of the JH sensor DNA. At this time, a linker sequence of several bases may be included between the signal DNA and the JH sensor DNA.

  The above DNA cloning method and JH sensor DNA preparation method can be achieved by known techniques in the art. For example, it may be prepared with reference to the method described in Green, M.R. and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. In this specification, as an example of JH sensor DNA preparation, the specific preparation method is described in Example 1 described later.

3. Juvenile hormone sensor expression vector 3-1. Overview A third aspect of the present invention is a juvenile hormone sensor expression vector (often referred to as a “JH sensor expression vector”) that includes the JH sensor DNA of the second aspect in an expressible state. . The JH sensor expression vector of the present invention can control the expression of the JH sensor. In addition, by introducing the transformant into a host arthropod, a JH in vivo kinetic monitoring system can be constructed by the JH in vivo kinetic monitoring method of the fifth aspect described later.

3-2. Structure In the present specification, the “JH sensor expression vector” refers to an expression system unit that can express the JH sensor DNA encoded in the vector in a state where it can be expressed and can induce the expression.

  As used herein, “expressible state” means that the JH sensor DNA is incorporated into an expression vector so that it can be expressed. Specifically, it means that the JH sensor DNA is placed under the control of a promoter in the JH sensor expression vector.

  The JH sensor expression vector of this embodiment includes a mother nucleus (core) vector, JH sensor DNA, and a promoter as essential constituent elements. In addition, terminator, enhancer, multiple cloning site, 5'UTR, 3'UTR, selection marker, insulator, transposon inverted terminal repeat sequence, cell membrane localization signal sequence, organelle localization signal sequence, etc. Can be included as a selection component. Hereinafter, each component of the JH sensor expression vector of this embodiment will be specifically described.

(1) Mother Nucleus (Core) Vector The mother nucleus vector constitutes the backbone of the JH sensor expression vector. Various vectors can be used as the mother nucleus vector. For example, a vector capable of autonomous replication such as a plasmid or Bacmid, a viral vector, or a vector capable of homologous recombination in a chromosome can be mentioned. As the mother nucleus vector, a vector that can replicate in the cells of the host organism to be introduced is generally selected. Further, the mother nucleus vector may be a shuttle vector that can replicate between other bacteria such as Escherichia coli and arthropods. Furthermore, protein expression vectors in which promoters, terminators, multicloning sites, selection markers, etc., described later in the mother nucleus vector have already been inserted, are commercially available from life science manufacturers, and these are used as JH sensor expression vectors. You can also

(2) JH sensor DNA
This JH sensor DNA is the JH sensor DNA described in the second embodiment. When a JH sensor is expressed as a secreted protein, it may be a JH sensor DNA in which a signal DNA is linked to the 5 ′ end. For example, when the JH sensor expression vector of the present invention is introduced into a host arthropod for monitoring the in vivo kinetics of JH in the fourth embodiment described later, it is necessary to use a JH sensor DNA to which signal DNA is linked. There is.

  When the signal DNA is not linked to the JH sensor, an ATG as an initiation codon is inserted into the 5 'end of the JH sensor DNA in order to start translation from a desired position after transcription. This is because JHBP constituting the JH sensor is a truncated peptide obtained by cleaving and removing the signal peptide, and does not contain an initiation methionine at its amino terminus.

(3) Promoter
The promoter contained in the JH sensor expression vector is an essential constituent element that controls the expression of the JH sensor in the JH sensor expression vector of the present invention. The type of the promoter is not particularly limited as long as it has a transcription control function in the host cell. A promoter known in the art may be used. In general, for example, an overexpressed promoter capable of overexpressing a target gene in a host, a constitutively active promoter capable of constant expression, and a time-specific expression that can be controlled depending on the stage of development of the host Known are active promoters, expression-inducible promoters whose expression can be freely controlled, and site-specific promoters that can be selectively expressed in specific tissues or specific sites of the host. Any promoter may be used and may be appropriately determined in consideration of the intended use of the JH sensor expression vector of the present invention.

(4) Terminator The terminator is a selective constituent element composed of a base sequence capable of terminating transcription when JH sensor DNA is expressed in the JH sensor expression vector of the present invention. There is no particular limitation as long as it is a sequence capable of terminating the transcription of JH sensor DNA transcribed by a promoter.

(5) Enhancer An enhancer is a regulatory element that controls a promoter, and is a selection component in the JH sensor expression vector of the present invention.

(6) Multicloning site The multicloning site is a cluster region including many restriction enzyme sites for cloning, and is a selection component in the JH sensor expression vector of the present invention. There are no particular restrictions on the type and number of restriction enzyme sites to be included in the base sequence constituting the multicloning site. In addition, there are no restrictions on the number of multicloning sites in the JH sensor expression vector and the location of the multicloning site, but the JH sensor expression vector should be placed at least within the control region of the promoter in a state in which JH sensor DNA can be expressed. It is preferable to keep it.

(7) 5'UTR and 3'UTR
5′UTR and 3′UTR are nucleic acid regions consisting of untranslated regions that do not themselves encode proteins or fragments thereof, or functional nucleic acids. The 3 ′ UTR can contain a poly A signal. It is a selection component in the JH sensor expression vector of the present invention. 5′UTR is located upstream of the start codon of JH sensor DNA, and 3′UTR is located downstream of the stop codon of JH sensor DNA.

(8) Selection marker The selection marker can function as a marker for confirmation indicating that the host holds the JH sensor expression vector of the present invention. It is a selection component of the JH sensor expression vector of the present invention. In general, a gene encoding an enzyme, a fluorescent protein, a chromogenic protein, a photoprotein, or the like is used as the selection marker. For example, drug resistance genes (for example, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, hygromycin resistance gene, spectinomycin resistance gene, chloramphenicol resistance gene or neomycin resistance gene), nutrient genes (for example, leucine, Biosynthesis genes of uracil, adenine, histidine, lysine or tryptophan), fluorescent or luminescent reporter genes (eg, luciferase, β-galactosidase, β-glucuronidase (GUS), or GFP), enzyme genes (neomycin phosphotransferase II (NPT II) ), Dihydrofolate reductase, etc.), and enzyme genes such as blasticidin S resistance gene. However, when a fluorescent or luminescent reporter gene is used as a selection marker, a fluorescent or luminescent spectrum that does not inhibit the activity of the chromophore involved in resonance energy transfer is selected. When the resonance energy transfer donating or accepting substance of the JH sensor is a fluorescent or photoprotein, it may be used as a selection marker. If necessary, a single JH sensor expression vector can include a plurality of the same or different selection markers.

(9) Insulator An insulator is a selective component in the expression vector of the JH sensor of the present invention, and can stably transcribe a gene sandwiched between its sequences without being affected by chromatin of surrounding chromosomes. It is a base sequence that can be controlled. For example, Drosophila gypsy sequences.

(10) Inverted terminal repeat sequence of transposon “Inverted terminal repeat sequence of transposon” is a selection structure that can be included when the JH sensor expression vector of the present invention is an expression vector capable of homologous recombination. Is an element. Inverted terminal repeats are usually used in pairs, for example, piggyBac, mariner, minos, etc. can be used as insect transposons (Shimizu, K. et al., 2000, Insect Mol. Biol., 9, 277-281; Wang W. et al., 2000, Insect Mol Biol 9 (2): 145-55).

3-3. Preparation of JH sensor expression vector The method for preparing the JH sensor expression vector of the present invention can also be achieved by a known technique in the art, similar to the method for preparing the JH sensor DNA of the second embodiment. For example, the method described in Green, MR and Sambrook, J., 2012 (described above) may be used. In this specification, a specific method for preparing a JH sensor expression vector is described as an example in Example 1 described later.

4). Transformant or its progeny 4-1. Outline The fourth aspect of the present invention is a transformant or a progeny thereof. The transformant of the present invention includes the JH sensor expression vector of the third aspect. If the transformant of the present invention is a unicellular organism such as Escherichia coli or yeast, it can be used as a JH sensor expression system. Moreover, if it is the transformant of arthropoda organism, it can be utilized as the transformant for in vivo kinetic monitoring of JH as described in the fifth aspect.

4-2. Configuration The transformant of the present invention includes the JH sensor expression vector of the third aspect in a cell. That is, the host transformed by introducing the JH sensor expression vector of the third aspect is the transformant of this aspect. Therefore, the transformant of this embodiment is composed of the host and the JH sensor expression vector of the third embodiment. Hereinafter, a specific description will be given.

(1) Host The host constituting the transformant of the present invention is not particularly limited as long as it is a cell or individual capable of expressing the JH sensor DNA included in the JH sensor expression vector. If the host is a prokaryotic cell, for example, Escherichia coli, Bacillus subtilis and the like can be mentioned. Moreover, if the host is a eukaryotic cell, cultured cells such as budding yeast (Saccharomyces cerevisiae), fission yeast (Schizosaccharomyces pombe), insect cells (for example, Sf9, Sf21, SF ⁺ , High-Five, BmN4) can be mentioned. . Furthermore, if the host is an individual, arthropods such as the above-mentioned insects, crustaceans, or sphenoid organisms may be used. A particularly preferred host for an individual is an insect.

(2) JH sensor expression vector The JH sensor expression vector included in the transformant of the present invention is the JH sensor expression vector according to the third embodiment. When the host is Escherichia coli, Bacillus subtilis, budding yeast, fission yeast, or cultured cells, the JH sensor DNA included in the JH sensor expression vector may or may not be linked to signal DNA. On the other hand, when the host is an individual of a multicellular organism, the JH sensor DNA included in the JH sensor expression vector preferably has a signal DNA linked thereto. The JH sensor expression vector may be integrated into the host genome.

(3) Progeny In this specification, “progeny” refers to an individual that is a first-generation offspring individual of a transformant and that retains the JH sensor expression vector of the third aspect in the cell. The number of generations of the progeny is not limited as long as the JH sensor expression vector of the third aspect is retained.

4-3. Method for producing transformant The transformant of the present invention is produced by introducing a JH sensor expression vector into a host. As a method for introducing the JH sensor expression vector, a known introduction method corresponding to the host to be introduced may be used. Examples of the method for introducing the vector into bacteria or yeast include a heat shock method, a method using calcium ions, and an electroporation method. These techniques are all known in the art and are described in various documents. For example, the method described in Green, MR and Sambrook, J., 2012 (described above) may be referred to. Examples of methods for introducing the vector into cells include the lipofectin method (PNAS, 1989, 86: 6077; PNAS, 1987, 84: 7413), electroporation method, calcium phosphate method (Virology, 1973, 52: 456-467), liposome method, DEAE-Dextran method and the like are preferably used. In addition, a known technique may be used as a method for introducing into an individual. For example, as a method for introducing a JH sensor expression vector into an insect individual, the method of Tamura et al. (Tamura T. et al., 2000, Nature Biotechnology, 18, 81-84) for introducing a gene expression vector into a silkworm is used. can do.

5). In vivo kinetic monitoring method for juvenile hormone 5-1. Outline | summary The 5th aspect of this invention is the in-vivo dynamics monitoring method of JH. The method of the present invention detects changes in the fluorescence spectrum in vivo based on FRET, using the individual into which the JH sensor according to the first aspect has been introduced or the transformant according to the fourth aspect. By this method, it is possible to monitor the dynamics of JH in vivo that could not be achieved by conventional JH detection and quantification methods.

In the present specification, “in vivo kinetics of JH” refers to a spatial position where JH exists in the living body, a positional amount of JH in the living body, and a temporal or positional change of JH.
As used herein, “living body” refers to a state in which an individual is alive.

5-2. Method The method of the present invention comprises a first embodiment and a second embodiment. Hereinafter, each embodiment will be described.

5-2-1. 1st Embodiment 1st Embodiment is a form which introduce | transduces the JH sensor as described in a 1st aspect into an individual | organism | solid, and performs in vivo dynamics monitoring using the individual | organism | solid. This embodiment includes an introduction process, a measurement process, and a JH detection process. Hereinafter, each step will be described.

(1) Introduction Step “Introduction step” is a step of introducing the JH sensor according to the first aspect into the arthropod of the subject.
The “subject” in this step refers to an arthropod individual that is subjected to the in vivo kinetic monitoring method of JH of the present invention. Arthropods are preferably insects, crustaceans or sphenoid organisms, and insects are more preferred.

  The method for introducing the JH sensor into the subject is not particularly limited. The easiest method is a method of injecting into a body fluid or tissue (including cells) using a syringe or the like. In addition, intracellular introduction via endocytosis and the introduction method described in Japanese Patent No. 5097412 or Japanese Patent No. 4885476 can be used.

(2) Measurement step The “measurement step” is a step of measuring fluorescence or luminescence generated by FRET or protein reconstruction derived from the JH sensor from the subject after the introduction step. As described above, the JH sensor of the present invention is based on the principle that the three-dimensional structure change of JHBP due to JH binding is regarded as luminescence or fluorescence due to FRET or protein reconstruction. By measuring the change in the emission or fluorescence spectrum, the presence and amount of JH in the living body can be indirectly detected and quantified.

  In this step, the emission or fluorescence spectrum generated by the binding of JH and JH sensor in the subject is detected and measured. The method for detecting and measuring luminescence or fluorescence from outside the living body is not limited, but for example, an in vivo bioimaging method can be used. Specifically, for example, Katz, MH et al., 2003, Cancer Res. 63: 5521-5525, Schmitt, CA et al., 2002, Cancer Cell, 1: 289-298, Katz, MH et al., 2003, J. Surg. Res., 113: 151-160 and the like, but are not limited thereto. Detection may be performed by a commercially available IVIS Imaging System (Caliper) or a similar device.

(3) JH detection step The "JH detection step" is a step of detecting JH based on the measurement value measured in the measurement step. The “measured value” is a numerical value obtained based on the measurement method of luminescence or fluorescence in the measurement step. The numerical unit varies depending on the measurement method. For example, if it is a method of detecting and measuring fluorescence, it will become a fluorescence intensity (Fluorescence intensity) (unit: arbitrary unit) etc. as a numerical unit.

  The detection of JH may be calculated as a relative value based on the magnitude of the measured value, a change with time, and / or a difference in measured value from a non-introduced individual that has not introduced the JH sensor. By analyzing the spatial position of the detected JH in the living body, changes over time, and positional changes, the dynamics of JH in the living body can be monitored.

5-2-2. 2nd Embodiment 2nd Embodiment is a form which performs in vivo dynamics monitoring using the transformant as described in a 4th aspect. This embodiment includes a transformant production step, a measurement step, and a JH detection step. Hereinafter, each step will be described.

(1) Transformation production process The "transformation production process" is a process for producing a transformant by introducing the JH sensor expression vector of the third aspect into a subject arthropod. This step is an unnecessary step when a transformant of the fourth form or a progeny thereof is available. In that case, the measurement may be performed from the next measurement step.

  The transformant in this step may be performed according to the method for producing a transformant described in the fourth aspect. For transformation of the host, a JH sensor expression vector including a JH sensor DNA having a signal DNA linked to the 5 'end is used. Moreover, the subject species and the species derived from the JHBP gene of the JH sensor DNA contained in the introduced JH sensor expression vector are not necessarily the same. This is because, as described above, the three-dimensional structure of JHBP is conserved among species, and even JH and JHBP between different species can bind to each other, resulting in a change in the three-dimensional structure of JHBP. Because. For example, the JHBP gene of the JH sensor DNA contained in the JH sensor expression vector may be derived from silkworms, and the host organism into which the JH sensor expression vector is introduced may be a crocodile that differs at the taxonomic level.

(2) Measurement step The “measurement step” is a step of measuring the fluorescence or luminescence generated by the FRET derived from the JH sensor expressed from the JH sensor expression vector of the transformant, or the luminescence or the reconstruction of the fluorescent protein. . Basically, it may be performed according to the measurement process of the first embodiment. Therefore, here, differences from the first embodiment will be described.

  This process may include an expression induction step. The “expression induction step” is a step of inducing the expression of JH sensor DNA inserted into the JH sensor expression vector. The necessity of this step is determined by the type of promoter that controls the expression of the JH sensor in the JH sensor expression vector. For example, this step is required when the promoter is an expression-inducible promoter. On the other hand, in the case of a constitutively active promoter, this step is unnecessary because the JH sensor is constantly expressed in the transformant. The expression induction method may be performed according to the nature of the expression inducible promoter. For example, in the case of a heat shock promoter, expression of the JH sensor can be induced by heating the transformant for a certain period of time.

(3) JH detection step The "JH detection step" is a step of detecting JH based on the measurement value measured in the measurement step. Basically, it may be performed according to the JH detection process of the first embodiment. Here, differences from the first embodiment will be described.

  The detection of JH can be calculated as a relative value based on the magnitude of the measurement value, a change with time, and / or a difference in measurement value from a non-transformant not containing the JH sensor expression vector of the third aspect. Good. By analyzing the spatial position of the detected JH in the living body, changes over time, and positional changes, the dynamics of JH in the living body can be monitored.

5-3. Effect In any of the conventional JH detection and quantification methods, it is necessary to destroy cells and tissues in detecting JH, and JH dynamics in vivo cannot be observed. According to the in vivo kinetic monitoring method of JH of the present invention, it is possible to monitor the in vivo kinetics of JH that could not be achieved by conventional methods.

6). 6. Method for searching juvenile hormone analogues 6-1. Outline A sixth aspect of the present invention is a JH analog search method. The method of the present invention is a method for screening JH analogs with high throughput. According to this method, a JH analog as a drug that effectively inhibits metamorphosis of insects and the like can be easily and quickly searched for and obtained.

  As used herein, “JH analog” refers to a substance that can disrupt the hormonal balance of JH and molting hormone (ecdysone) in arthropods and consequently inhibit molting and / or metamorphosis. For example, a substance showing an action similar to JH, or a substance showing an action as an antagonist that binds to JHBP or JH receptor and antagonizes the function of JH can be mentioned. JH analogs are often structurally similar to JH, but need not necessarily be structural analogs of JH as long as they have the above action.

  Since mammals including humans do not have JH or molting hormone, JH analogs are not toxic to mammals or very low. Therefore, JH analogs can be effective insecticides that are highly safe for mammals. Currently, JH analogs such as metoprene, phenoxycarb, and pyriproxyfen are used as insecticides.

6-2. Method The method of the present invention includes a mixing step, a measuring step, and a selecting step.
(1) Mixing step The “mixing step” is a step of mixing the JH sensor according to the first aspect and a candidate substance.

  A “candidate substance” is a substance that can be a candidate for a target JH analog. Although it may be a low molecular compound, a nucleic acid, or a peptide, it is preferably the same low molecular compound as JH. Low molecular weight compounds that are structurally similar to JH are more preferred. The candidate substance may be prepared by any known synthesis method. For example, it can be synthesized by an organic chemical reaction.

The method of the present invention may be performed in either an in vitro system or an in vivo system.
When performing the method of the present invention in an in vitro system, a JH sensor is prepared in advance. The JH sensor can be extracted from a transformant of a unicellular organism such as Escherichia coli or yeast or a cultured cell such as an insect cell that includes the JH sensor expression vector of the present invention by a known protein expression method. For specific methods for culturing Escherichia coli, yeast, cultured cells, etc., protein expression methods, and protein extraction methods, the methods described in Green, MR and Sambrook, J., 2012 (described above) may be referred to. If the transformant includes a JH sensor expression vector containing a JH sensor gene having a signal DNA, the expressed JH sensor is secreted into the culture solution, and the culture supernatant is recovered. You can get a JH sensor. The culture supernatant containing the JH sensor can be collected using a known protein separation and purification technique, for example, gel filtration, ion exchange chromatography, or affinity chromatography, if necessary.

  The JH sensor and the candidate substance are mixed in an appropriate buffer or reaction solution. Tris, HEPES, phosphate buffer, etc. may be used as the buffer. The mixing ratio of the two is such that the JH sensor: candidate substance is 1: 0.1 to 1: 1000. Mixing can be achieved by gently stirring the mixed solution with a stir bar or a stir bar. As a positive control, it is desirable to mix the JH sensor and JH under the same conditions.

  When the method of the present invention is performed in an in vivo system, a transformant transformed with a JH sensor expression vector containing a JH sensor gene having no signal DNA is used as it is.

  Mixing can be achieved by adding the candidate substance in a culture solution of the transformant or by collecting the transformant, washing it and then suspending it in an appropriate buffer or the like.

(2) Measurement step The “measurement step” is a step of measuring the fluorescence or luminescence generated by the luminescence or fluorescence resonance energy transfer of the JH sensor or the reconstruction of the fluorescent protein.

  In this step, the emission or fluorescence spectrum generated by the binding between the JH sensor and the candidate substance is detected and measured. The measuring method is not particularly limited. For example, a known method using a fluorescence plate reader or a fluorescence spectrophotometer can be used.

(3) Selection Step The “selection step” is a step of selecting candidate substances based on the measurement values obtained in the measurement step as JH analogs.

  The method for selecting a candidate substance based on a measured value is not limited. For example, a method of selecting a candidate substance based on a significant difference from a measured value of a negative control in which no candidate substance is mixed in the mixing step, or a concentration-dependent measured value of a candidate substance. A selection method based on the increase in

  In the selection method based on the significant difference, for example, when the measured value when the candidate substance is mixed is compared with the measured value when the candidate substance is not mixed, the candidate substance is selected when there is a statistically significant difference between the two. It may be selected as a JH analog. “Statistically significant” includes, for example, the case where the risk value (significance level) of the obtained value is small, specifically, the case of p <0.05, p <0.01 or p <0.001. Here, “p” or “p value” indicates the probability that an assumption is accidentally correct in the distribution assumed by the statistic in the statistical test. Therefore, the smaller the “p” or “p value”, the closer the assumption is to true. “Statistically significant difference” means that there is a significant difference between the measured value when the candidate substance is mixed and the measured value when not mixed when statistically processed. Say. The test method for statistical processing is not particularly limited as long as a known test method capable of determining the presence or absence of significance is appropriately used. For example, Student's t test or multiple comparison test can be used.

  In the selection method based on the concentration-dependent increase in the measured value of the candidate substance, for example, the candidate substances are mixed at different concentrations in the mixing step. When the measured value at each concentration increases in a concentration-dependent manner, the candidate substance may be selected as a JH analog.

<Example 1: Construction of expression vector for JH sensor>
The JH sensor expression vector of the present invention was constructed.
(1) Construction of mTFPmVenus_pRSET-A (Kpn) _ver1.2 Using pRSET-A-BFP (Life technologies), an E. coli protein expression vector, as a mother nucleus, BFP ORF (open reading frame) contained in the vector “MTFPmVenus_pRSET-A (Kpn) _ver1.2” was constructed by replacing with FRET chromophore ORF. As the chromophore ORF of FRET, cyan fluorescent protein mTFP1 ORF consisting of the base sequence shown in SEQ ID NO: 11 and yellow fluorescent protein mVenus ORF consisting of the base sequence shown in SEQ ID NO: 13 were used.

  Specifically, first, PCR was performed using primer pair mVenus-f1 (SEQ ID NO: 16) and pRSETA-r1 (SEQ ID NO: 17) to which restriction enzyme recognition sequences were added using mVenus-pRSET-B as a template. . As thermostable polymerases, polymerases with a calibration function PrimeSTAR HS and PrimeSTAR Max (TaKaRa Bio) were used. The reaction conditions were 94 ° C. for 30 seconds, 58 ° C. for 30 seconds and 72 ° C. for 90 seconds, and 35 cycles were performed with a thermal cycler (MyCycler; Bio-Rad). The PCR product after the reaction is purified using an ethanol precipitation and PCR purification kit (NucleoSpin Gel and PCR Clean-up; MACHEREY-NAGEL), and then the PCR product and pRSET-A-BFP are treated with Kpn1 and Hind III. did. The target DNA strand was purified using ethanol precipitation and PCR purification kit (Nucleo Spin Gel and PCR Clean-up; MACHEREY-NAGEL), and then ligated by ligation enzyme reaction using Mighty Mix (TaKaRa Bio). The reaction conditions were in accordance with the attached protocol. Escherichia coli K12 strain DH-5a was transformed with the reaction solution after the ligation reaction, and plasmid mVenus-pRSET-A was extracted from the obtained transformant using Plasmid DNA Mini-PrepKit (Life technologies). Next, in order to insert mTFP1 into the 5 'end of Mvenus, mTFP1 ORF was amplified by PCR using mTFP-pGEMT as a template and using mTFP1_f1 (SEQ ID NO: 18) and mTFP1_r1 (SEQ ID NO: 19). The obtained PCR product and mVenus-pRSET-A were treated with restriction enzymes with BamHI and KpnI. Purification of the target DNA strand, ligation reaction, and transformation method were in accordance with the method for preparing mVenus-pRSET-A. The final mTFPmVenus_pRSET-A (Kpn) obtained was sequenced with ABI Prism 3730 (Life technologies) using BigDye Terminator v3.1 cycle sequencing kit (Life technologies). Sequence analysis and assembly were analyzed with GENETYX-MAC ver. 16 software (GENETYX, Tokyo, Japan). A plasmid map of the completed mTFPmVenus_pRSET-A (Kpn) _ver1.2 is shown in FIG. 3A.

(2) Construction of mTFPmVenus_pRSET-A (RV) _ver1.2
In mTFPmVenus_pRSET-A (Kpn) _ver1.2, the restriction enzyme recognition sequence between mTFPm ORF and Venus ORF is only KpnI (see FIG. 3A). Therefore, in constructing a JH sensor, convenience at the time of inserting a JHBP gene is low. Therefore, mTFPmVenus_pRSET-A (RV) _ver1.2 with restriction enzyme recognition sequences (XhoI, EcoRV, KpnI) added was constructed by Inverse PCR method. The primer pair shown by sequence number 20 and sequence number 21 was used as a primer for nucleic acid amplification. A plasmid map of the completed mTFPmVenus_pRSET-A (RV) _ver1.2 is shown in FIG. 3B.

(3) Construction of JH sensor
A FRET-JHBP chimeric protein expression vector containing FRET-JHBP-TypeA to TypeC was constructed. FRET-JHBP-TypeA to TypeC (FIG. 4) encode chimeric proteins having different insertion positions of mTFP and mVenus into JHBP, respectively. Of these, FRET-JHBP-TypeA has the configuration of the JH sensor of the present invention.

(i) Construction of FRET-JHBP-TypeC For mature JHBP ORF, silkworm mature JHBP II ORF (BmJHBP II) consisting of the base sequence shown in SEQ ID NO: 3 was used (Vermunt et al., 2001. Insect Mol. Biol. 10: 147). PCR was performed using JHBPII-pGEX2T as a template and primer JHBP_f1 (SEQ ID NO: 22) with Xho I added to the 5 ′ end and primer JHBP_r1 (SEQ ID NO: 23) added with Kpn I on the 3 ′ end. The PCR product was purified and then cleaved with XhoI and KpnI to obtain a BmJHBP II DNA fragment (XhoI / KpnI). Subsequently, mTFPmVenus_pRSET-A (RV) _ver1.2 was cleaved with XhoI and KpnI to incorporate a BmJHBP II DNA fragment (XhoI / KpnI). PCR conditions and ligation reaction conditions were performed according to the method (1). As a result, FRET-JHBP-TypeC was obtained in which mTFP ORF was linked to the 5 ′ end of BmJHBP II ORF and mVenus ORF was linked to the 3 ′ end (FIG. 4).

(ii) Construction of FRET-JHBP-TypeA
From the three-dimensional structure information of JHBP at the time of JH binding and detachment (Suzuki et al., 2011), an amino acid region in which FRET efficiency is increased by inserting a fluorescent protein into JHBP was estimated. One of the candidate regions is the 10th to 15th amino acids in mature JHBP, an mTFP ORF is inserted between the 11th lysine codon (L11) and the 12th glycine codon (G12) of BmJHBP, and the mVenus ORF is replaced with JHBP ORF. FRET-JHBP-TypeA (FIG. 4) linked to the 3 ′ end of was constructed.

  FRET-JHBP-TypeA was constructed from two DNA fragments of an N-terminal region and a C-terminal region bounded by positions 33 and 34 of BmJHBP ORF, which is the insertion position of mTFP1 ORF.

  First, an N-terminal region was prepared by PCR primer extension reaction in which the base sequence of 1st to 33rd position encoding the amino acid residue of 1st to 11th position of BmJHBP was added to the 5 'end side of mTFP1 ORF. Using FRET-JHBP-TypeC as a template, AJH_Ff1 (SEQ ID NO: 24), AJH_Ff2 (SEQ ID NO: 25) and AJH_Ff3 (SEQ ID NO: 26) are used for the forward primer, and AJH_Fr1 (SEQ ID NO: 27) is used for the reverse primer. It was. A pBluescript II (sk) sequence and a BamHI recognition sequence functioning as a GeneArt recognition sequence are added to the 5 'end of AJH_Ff3. Further, AJH_Fr1 includes a base sequence on the 3 ′ end side of the N-terminal region and the 5 ′ end side of the C-terminal region, and is designed to overlap (complement) with AJH_Rf1 described later. PCR reaction conditions were set to 35 cycles using a thermal cycler (MyCycler; Bio-Rad) with 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 72 ° C. for 90 seconds as one cycle. The PCR product obtained after the reaction was cloned directly into the pCR-Blunt TOPO of Zero Blunt (registered trademark) PCR Cloning Kit (Life technologies) according to the attached protocol, and then BigDye Terminator v3.1 cycle sequencing kit (Life technologies) The base sequence of the PCR product was confirmed.

On the other hand, the C-terminal region in which Mvenus ORF is added to the 3 ′ end of the base sequence from the 33rd position to the 675th position encoding the amino acid residues from the 12th position to the 225th position of BmJHBP is the template using the aforementioned FRET-JHBP-TypeC as a template. Prepared by PCR. AJH_Rf1 (SEQ ID NO: 28) was used as the forward primer, and AJH_Rr1 (SEQ ID NO: 29) was used as the reverse primer. As described above, AJH_Rf1 includes a base sequence on the 3 ′ end side of the N-terminal region and the 5 ′ end side of the C-terminal region, and is designed to be complementary to AJH_Fr1. In addition, a pBluescript II (sk) sequence and a Hind III recognition sequence functioning as a GeneArt recognition sequence are added to the 5 ′ end of AJH_Rr1. The PCR product obtained after the reaction was directly cloned by the same method as that for the N-terminal region, and the base sequence of the PCR product was confirmed using BigDye Terminator v3.1 cycle sequencing kit (Life technologies).
Subsequently, using N-terminal region and C-terminal region as templates, forward primer AJH-Ff3 (SEQ ID NO: 26) that binds to 5′-terminal GeneArt recognition sequence of N-terminal region and 3′-terminal GeneArt recognition sequence of N-terminal region Reverse primer AJH-Fr1 (SEQ ID NO: 27) that binds to C5, the 5 ′ end GeneArt recognition sequence of the C-terminal region, and the forward primer AJH-Rf1 (SEQ ID NO: 28) that binds to the 5′-end GeneArt recognition sequence of the C-terminal region PCR was performed using the reverse primer AJH_Rf1 (SEQ ID NO: 29) that binds to FRET-JHBP-TypeA by GeneArt Seamless cloning (Life technologies). After cloning the PCR product containing the full-length fragment directly into pBluescript II (sk), determine the base sequence of the PCR product using the BigDye Terminator v3.1 cycle sequencing kit (Life technologies) and place it at a specific position in the JHBP ORF. It was confirmed that mTFP1 ORF was inserted seamlessly. PRSET-A-BFP after excising the full length fragment of FRET-JHBP-TypeA from pBluescript II (sk) by cutting with Bam HI and Hind III, and then removing the BFP ORF by cutting with Bam HI and Hind III (Life technologies) inserted into the Bam HI / Hind III restriction enzyme recognition sequence. The obtained FRET-JHBP-TypeA chimeric protein expression vector was designated as “FRET-JHBP-TypeA_pRSE Ta”. The nucleotide sequence of FRET-JHBP-TypeA_pRSE Ta is shown in SEQ ID NO: 30. As described above, this expression vector has the configuration of the JH sensor expression vector of the present invention.

(iii) Construction of FRET-JHBP-TypeB
As other deduced amino acid regions that increase FRET efficiency, select the asparagine codon at position 137 (N137) and glycine codon at position 138 (G12) of mature BmJHBP, insert mVenus ORF between these amino acid residues, and FRET-JHBP-TypeB (FIG. 4) was constructed by linking mTFP ORF to the 5 ′ end of BmJHBP.

  Like FRET-JHBP-TypeA, FRET-JHBP-TypeB is also constructed from three DNA fragments, the N-terminal region and C-terminal region of the BmJHBP ORF, which is the insertion position of mTFP1 ORF, at the 137th and 138th positions, and mVenus ORF. did.

  The basic method was based on the preparation method of FRET-JHBP-TypeA. First, the N-terminal region is the FRET-JHBP-TypeC described later as a template, and the forward primer Ave_f1 (SEQ ID NO: 31) containing the base sequence of the 5 ′ end region of mTFP1 ORF and the amino acid residues at positions 130 to 137 of BmJHBP It was prepared by PCR using a reverse primer BJH-Fr1 (SEQ ID NO: 32) containing a base sequence complementary to the encoded base sequence at positions 388 to 411. A pBluescript II (sk) sequence and a BamHI recognition sequence functioning as a GeneArt recognition sequence are added to the 5 'end of Ave_f1. Furthermore, BJH_Fr1 includes a base sequence on the 3 ′ end side of the N-terminal region and the 5 ′ end side of mVenus, and is designed to overlap (complement) with BJH_vef1, which will be described later. The PCR conditions were in accordance with the method for preparing FRET-JHBP-TypeA, and the obtained PCR product was directly cloned in the same manner as the N-terminal region of FRET-JHBP-TypeA, then BigDye Terminator v3.1 cycle sequencing kit ( Life technologies) was used to confirm the base sequence of the PCR product.

  Next, mVenus ORF uses FRET-JHBP-TypeC, which will be described later, as a template, forward primer BJH_vef1 (SEQ ID NO: 33) containing the base sequence of the 5 ′ end region of mVenus ORF and the base sequence of the 3 ′ end region of mVenus ORF. It was prepared by PCR using a reverse primer BJH_ver1 (SEQ ID NO: 34) containing a complementary base sequence. BJH_vef1 includes a base sequence on the 3 ′ end side of the N-terminal region and the 5 ′ end side of the mVenus ORF, and is designed to overlap (complement) with BJH_Fr1 described above. BJH_ver1 includes a base sequence complementary to the base sequence on the 3 ′ end side of the mVenus ORF and the 5 ′ end side of the C terminal region, and is designed to overlap (complement) with BJH_Rr1 described later. PCR conditions were carried out according to the FRET-JHBP-TypeA preparation method, and the obtained PCR product was directly cloned in the same manner as the N-terminal region of FRET-JHBP-TypeA, and then BigDye Terminator v3.1 cycle sequencing The base sequence of the PCR product was confirmed using kit (Life technologies).

  Finally, the C-terminal region is a forward primer BJH_Rf1 (SEQ ID NO: 35) containing the above-mentioned FRET-JHBP-TypeC as a template and a base sequence from positions 412 to 429 encoding the amino acid residues at positions 138 to 143 of BmJHBP. ) And a reverse primer BJH_Rr1 (SEQ ID NO: 36) containing a base sequence complementary to the base sequence on the 3 ′ end side of BmJHBP ORF. BJH_Rf1 includes a base sequence on the 3 ′ end side of the mVenus ORF and the 5 ′ end side of the C-terminal region, and is designed to overlap (complement) with BJH_ver1 described above. BJH_Rr1 includes a base sequence complementary to the base sequence on the 3 ′ end side of the C-terminal region, and a pBluescript II (sk) sequence and a Hind III recognition sequence functioning as a GeneArt recognition sequence are added to the 5 ′ end. ing. After cloning the PCR product obtained after the reaction directly in the same way as the N-terminal region of FRET-JHBP-TypeA, the base sequence of the PCR product was confirmed using BigDye Terminator v3.1 cycle sequencing kit (Life technologies) .

  PCR was performed using the N-terminal region, mVenus ORF, and C-terminal region as templates to prepare a full-length fragment of FRET-JHBP-TypeB. After cloning the PCR product containing the full-length fragment directly into pBluescript II (sk), determine the base sequence of the PCR product using the BigDye Terminator v3.1 cycle sequencing kit (Life technologies) and place it at a specific position in the JHBP ORF. It was confirmed that mTFP1 ORF was inserted seamlessly. PRSET-A-BFP after excising the full-length fragment of FRET-JHBP-TypeB from pBluescript II (sk) by digestion with Bam HI and Hind III and then removing the BFP ORF by digestion with Bam HI and Hind III (Life technologies) inserted into the Bam HI / Hind III restriction enzyme recognition sequence. The obtained FRET-JHBP-TypeB chimeric protein expression vector was designated as “FRET-JHBP-TypeB_pRSE Ta”. The nucleotide sequence of FRET-JHBP-TypeB_pRSE Ta is shown in SEQ ID NO: 37.

<Example 2: Production of transformant and expression and purification of JH sensor>
A transformant was prepared by introducing the three types of gene expression vectors prepared in Example 1 into a host, and expression induction and purification of the FRET-JHBP chimeric protein encoded by each gene expression vector were performed.

Each of the FRET-JHBP chimeric protein expression vectors was introduced into E. coli BL21 (DE3) strain (Novagen) based on a conventional method. After culturing the obtained transformant in LB medium at 37 ° C., when the turbidity (OD ₆₀₀ ) reached 0.4 to 0.6, the culture temperature was lowered to 16 ° C., and a final concentration of 1 mM IPTG was added. Protein expression induction treatment was performed for ˜16 hours. Thereafter, E. coli was collected, the cells were resuspended in PBS (pH 7.5), and subjected to ultrasonic disruption treatment in an ice bath at an ultrasonic output of 70 to 100% for 20 to 30 minutes. The cell disruption solution was centrifuged at 40,000 × g for 30 minutes, and then the soluble fraction was collected and subjected to affinity purification with 5 mL of HisTrap HP (GE healthcare). The target product was eluted with a buffer containing 400 mM imidazole (50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 400 mM imidazole), and the contaminant was buffered with 20 mM imidazole (20 mM Tris- Elution was performed with HCl (pH 8.0), 200 mM NaCl, and 20 mM imidazole. Furthermore, the fraction containing the chimeric protein was purified by gel filtration chromatography using HiLoad 26/60 Superdex 200 prep grade (GE healthcare). 10 mM Tris-HCl (pH 7.5), 150 mM NaCl was used as a buffer. Final purification was confirmed by SDS-PAGE. The concentration of the chimeric protein obtained by expression from each gene expression vector was determined by UV measurement.

<Example 3: Fluorescence measurement of FRET-JHBP chimeric protein>
The fluorescence intensity in the presence of JH was measured for each FRET-JHBP chimeric protein prepared and purified in Example 2.

  Under the buffer conditions of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% ethanol, the FRET-JHBP chimeric protein (5 μM: TypeA or TypeB; 2 μM: TypeC) is mixed with JH III (Sigma-Aldrich) as a substrate. ) At a final concentration of 10 μM and incubated for 1 hour at 16 ° C. in the dark. As a control, a sample without JH III was prepared in the same manner. After the reaction, fluorescence measurement was performed using a fluorescence spectrophotometer F-4500 (HITACHI) under conditions of an excitation wavelength of 450 nm, a scan region of 480 to 550 nm, and a temperature of 20 ° C. The ratio between the fluorescence peak of mTFP1 (494 nm) and the fluorescence peak of mVenus (527 nm) was measured, and the change in fluorescence intensity with the JH concentration was determined.

  The results are shown in FIG. A is the result when FRET-JHBP-TypeA is used, B is FRET-JHBP-TypeB, and C is FRET-JHBP-TypeC. In FRET-JHBP-TypeA having the configuration of the JH sensor of the present invention, FRET was observed in the presence of JH III (10 μM). However, in FRET-JHBP-TypeB and FRET-JHBP-TypeC, the change in fluorescence intensity in the presence of JH III was almost the same pattern as that in the absence of JH III (0 μM), and FRET could not be detected. From this result, it is clear that in order to detect the binding of JHBP and JH using FRET or BRET, it does not function unless a fluorescent protein or the like is inserted in a limited specific portion shown in the present invention. It became.

<Example 4: Construction of high-stability FRET-JHBP sensor and fluorescence measurement thereof>
From the results of Example 3, it was revealed that FRET-JHBP-TypeA functions as a JH sensor. However, FRET-JHBP-TypeA has a problem that FRET is unstable. Therefore, we developed a JH sensor with high stability based on the TypeA sequence.

  In general, the fluorescent protein C-terminal is rich in hydrophilicity, does not contribute to the three-dimensional structure of the fluorescent protein, and is known not to affect fluorescence. It is stable by deleting and inserting amino acid residues of these linker sequences. We thought that expression of sensor protein was possible. Therefore, a construct was constructed in which the C-terminal of mTFP1 inserted into JHBP was deleted and inserted. Although the number of amino acid residues that can be trimmed at the C-terminus of mTFP1 has not been clarified, variant proteins such as CFP and GFP were able to trim C-terminal 9 amino acid residues. When homology with mTFP1 was confirmed, it was found that the C-terminal sequence GMDELYK was consistent with other fluorescent protein sequences. Therefore, in the FRET-JHBP-TypeA chimeric protein, mTFP1-dX (X), in which the construct trimmed by 1 amino acid residue from the 3 ′ end of the mTFP1 ORF, that is, the base (3-15 bases) coding for 1-5 amino acids was deleted. = An integer of 1 to 5) was prepared from FRET-JHBP-TypeA_pRSE Ta by site directed mutagenesis method. The obtained FRET-JHBP-TypeA chimeric protein expression vector was designated as “FRET-JHBP-TypeAdX_pRSE Ta” (X = 1 to 5).

  In addition, an amino acid residue was inserted into the C-terminal of the fluorescent protein to improve the stability of the sensor protein by enhancing the operability of the linker part between the JHBP sensor part and the fluorescent protein. The amino acid residue to be inserted is selected from glycine (G), which can take a variety of three-dimensional structures, because it has the largest conformation space among 20 types of amino acids, and the number of inserted amino acids is 1 or 2. It was. In the FRET-JHBP-TypeA chimeric protein expression vector, mTFP1-in (n = 1 or 2) with 1 or 2 glycine (Gly) residue-encoding bases added to the 3 ′ end of the mTFP1 ORF ) Was prepared from FRET-JHBP-TypeA_pRSE Ta by the site directed mutagenesis method. The obtained FRET-JHBP-TypeA chimeric protein expression vector was designated as “FRET-JHBP-TypeAin_pRSE Ta” (n = 1 or 2).

  Furthermore, in the FRET-JHBP-TypeC chimeric protein expression vector, the ORF of mTFP1-d5 in which the base (15 bases) encoding 5 amino acids was deleted from the 3 'end of the mTFP1 ORF was replaced with the ORF of mTFP1, and mVenus ORF of mVenus-d5 from which 5 amino acid coding bases (15 bases) have been deleted from the 3 'end of mVenus ORF between lysine codon (K83) at position 83 and alanine codon (A84) at position 84 (K83 / A84) ) To construct a FRET-JHBP-TypeCd5 chimeric protein expression vector “FRET-JHBP-TypeCd5_pRSE Ta”.

  Finally, insert the mTFP1-d5 ORF between the leucine codon at position 11 (L11) and the glycine codon at position 12 (G12) (L11 / G12), and a base encoding 4 amino acids from the 3 'end of the mVenus ORF Inserting the ORF of mVenus-d4 with (12 bases) deleted between the asparagine codon (N137) at position 137 and the glycine codon (G138) at position 138 (N137 / G138), FRET-JHBP-TypeDd5 chimeric protein expression vector “FRET-JHBP-TypeDd5_pRSE Ta” was constructed.

According to the method described in Example 3, the fluorescence intensity in the presence of JH in each FRET-JHBP chimeric protein was measured by an in vitro assay.
The results are shown in Table 1.

  In the table, the FRET value indicates the difference in the fluorescence intensity ratio in the presence and absence of JHIII (10 μM). From the FRET value, FRET-JHBP chimeric protein that can function as a JH sensor was evaluated with “◯”, and FRET-JHBP chimeric protein that could not function was evaluated with “×”.

  From the above results, it can be seen that the chimeric protein based on FRET-JHBP-TypeA, that is, when the insertion position of one fluorescent protein is L11 / G12 and the insertion position of the other fluorescent protein is C-terminal, It has been demonstrated that it can function as a sensor. Among them, the JH sensors TypeAd4 and TypeAd5 from which mTFP1-d4 and -d5 were removed were found to be JH biosensors with high FRET efficiency and high stability.

  However, if one fluorescent protein is inserted at the N-terminal and / or the other fluorescent protein is inserted at a position other than the C-terminal, it is clear that the FRET-JHBP chimeric protein cannot function as a JH sensor. It became.

<Example 5: Detection of JH or JH analog by JH sensor>
In Examples 3 and 4, JH III was used as JH. It is verified that the JH sensor of the present invention can detect JH or JH analog other than JH III.

  Using TypeAd5 prepared in Example 4 as a JH sensor, various JHs (JH I (SciTech), JH II (SciTech), JH III (Sigma-Aldrich), or MF (Echelon Biosciences))) or JH analogs (methoprene) The fluorescence intensity of FRET was measured over time in the presence of (AccuStandard) or Metoprenic acid (Sigma-Aldrich). Oleic acid (Sigma-Aldrich) was used as a negative control for JH.

  The basic measurement method was in accordance with the method described in Example 3. The amount of each JH or JH analog added was 1.38 μM and 10 μM, and samples without JH were prepared under the same conditions for control purposes. The fluorescence measurement time was 1 hour, 2 hours, 3 hours, and 6 hours after the addition of various JHs or JH analogs.

  The results are shown in FIG. Both JH and JH analogs were able to detect FRET by the TypeAd5 JH sensor. Moreover, the higher the concentration of JH or JH analog, the higher the detected fluorescence intensity. From the above results, it was proved that the JH sensor of the present invention can detect not only JH III but also various JHs and JH analogs.

<Example 6: JH sensor quantification>
It was confirmed that JH or JH analog in a sample can be quantified by the JH sensor of the present invention.
In a 96-well microplate (Falcon # 3072), 198 μL of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCL so that the final concentration of the JH sensor (FRET-JHBP-TypeAd5) prepared in Example 4 is 1 μM. In addition, 0.1 nM to 1 × 10 ⁷ nM JH (JH I (SciTech), JH II (SciTech), JH III (Sigma-Aldrich), MF (Echelon Biosciences))) or JH analog (methoprene) 2 μL of an acid solution (Sigma-Aldrich) in ethanol was added to each well for each concentration and allowed to stand at room temperature (about 25 ° C.) for 1 hour in the dark. After the reaction, the fluorescence intensity at excitation wavelength 450 nm, measurement wavelength 480, and 535 nm in each well was measured with a fluorescence plate reader (Perkin-Elmer ARVO MX; 1420 Multilabel Counter). A fluorescence intensity ratio of 535 nm / 480 nm was calculated.

  The results are shown in FIG. As shown in this figure, concentration-dependent FRET of JH and JH analogs was observed, and it was revealed that various ligands can be quantified by the JH sensor of the present invention.

Claims (18)

In the truncated amino acid sequence excluding the signal peptide of juvenile hormone binding protein in Lepidoptera insects,
When the amino acid residue at the amino terminus of the truncated amino acid sequence is the 1st position, any one amino acid residue at positions 10 to 15 accepts the resonance energy donating substance and the excitation energy of the resonance energy donating substance A juvenile consisting of a protein that is modified by one of the resonance energy accepting substances and the amino acid residue at the carboxyl terminus of the truncated amino acid sequence is modified by the other of the resonance energy donating substance and the resonance energy accepting substance. Young hormone sensor.   The juvenile hormone sensor according to claim 1, wherein the resonance energy donating substance is a luminescence or fluorescence resonance energy donating substance. In the truncated amino acid sequence excluding the signal peptide of juvenile hormone binding protein in Lepidoptera insects,
When the amino acid residue at the amino terminus of the truncated amino acid sequence is position 1, the resonance energy donating substance and excitation of the resonance energy donating substance between two consecutive amino acid residues within the range of positions 10 to 15 A juvenile polypeptide comprising a fusion polypeptide in which one of resonance energy accepting substances that receive energy is linked, and the other of the resonance energy donating substance and the resonance energy accepting substance is linked to the carboxyl terminus of the truncated amino acid sequence. Young hormone sensor.   The juvenile hormone sensor according to claim 3, wherein the resonance energy donating substance and / or the resonance energy accepting substance is a peptide.   The juvenile hormone sensor according to claim 4, wherein the peptide is a luminescent or fluorescent peptide. In the truncated amino acid sequence excluding the signal peptide of juvenile hormone binding protein in Lepidoptera insects,
When the amino acid residue at the amino terminus of the truncated amino acid sequence is defined as the first position, the amino terminal side of the luminescent or fluorescent protein fragment divided into two between two consecutive amino acid residues within the range of 10 to 15 positions A juvenile hormone sensor comprising a fusion polypeptide comprising an amino acid sequence linked and a carboxyl-terminal amino acid sequence of a luminescent or fluorescent protein fragment divided into two at the carboxyl terminus of the truncated amino acid sequence.   The juvenile hormone sensor according to any one of claims 3 to 6, wherein the distance between the two consecutive amino acid residues is between the 11th and 12th amino acid residues. The juvenile hormone sensor according to any one of claims 3 to 7, wherein the truncated amino acid sequence is any amino acid sequence represented by (a) to (c).
(A) an amino acid sequence represented by SEQ ID NO: 2, 4, 5 or 8;
(B) an amino acid sequence in which one or more amino acid residues are deleted, substituted or added in any one of the amino acid sequences described in (a), or (c) any one of the amino acid sequences described in (a) Amino acid sequence having 90% or more amino acid identity to the amino acid sequence   DNA which codes the fusion polypeptide which comprises the juvenile hormone sensor as described in any one of Claims 4-8.   A juvenile hormone sensor expression vector comprising the DNA according to claim 9 in an expressible state.   A transformant obtained by introducing the juvenile hormone sensor expression vector according to claim 10 into a host cell or host organism, or a progeny thereof. A method for monitoring the dynamics of juvenile hormone in an arthropod,
Introducing the juvenile hormone sensor according to any one of claims 1 to 8 into an arthropod of the subject;
A step of measuring luminescence or fluorescence generated by reorganization of luminescence or fluorescent protein in the subject subject to variation of resonance energy transfer in the subject, and detecting juvenile hormone based on the measurement value measured in the measurement step Said method comprising the steps.   13. The method of claim 12, wherein the fluctuating energy mutation is luminescence or fluorescence. A method for monitoring the dynamics of juvenile hormone in an arthropod,
Introducing the juvenile hormone sensor expression vector according to claim 10 into a subject arthropod to produce a transformant;
A step of measuring luminescence or fluorescence generated by reconstitution of luminescence or fluorescent protein, or an energy mutation that varies due to luminescence or fluorescence resonance energy transfer derived from a juvenile hormone sensor expressed from a juvenile hormone sensor expression vector in the transformant And the method comprising the step of detecting juvenile hormone based on the measurement value measured in the measurement step.   15. The method of claim 14, wherein the fluctuating energy mutation is luminescence or fluorescence. A method of searching for juvenile hormone analogs that are structurally similar to arthropod juvenile hormone,
Mixing the juvenile hormone sensor according to any one of claims 1 to 8 with a candidate substance,
A step of measuring fluorescence generated by energy variation due to resonance energy transfer of the juvenile hormone sensor or reconstitution of luminescent or fluorescent protein, and a juvenile hormone analog based on the measurement value obtained in the measurement step The method comprising the step of selecting as   The method of claim 16, wherein the fluctuating energy mutation is luminescence or fluorescence.   The method according to claim 16 or 17, wherein the selecting step is based on a significant difference from a measured value of a negative control in which no candidate substance is mixed in the mixing step, or an increase in a concentration-dependent measured value of the candidate substance.

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