JP2023109567A - Lipid membrane structure for odorant detection, lipid membrane sensor for odorant detection, odorant detection method, and method for producing lipid membrane structure - Google Patents

Abstract

A novel structure capable of detecting an odorant to be detected with high sensitivity is provided. SOLUTION: A lipid membrane structure for detecting an odorant of the present invention includes a lipid membrane, an olfactory receptor, and a co-receptor, the lipid membrane has a bag-like structure, and to the olfactory receptor and the co-receptor, wherein the olfactory receptor comprises the Or42a receptor and/or the Or43b receptor, and the co-receptor comprises an Orco family receptor. [Selection diagram] Fig. 1

Description

The present invention relates to an odorant-detecting lipid membrane structure, an odorant-detecting lipid membrane sensor, an odorant-detecting method, and a method for producing a lipid membrane structure.

Methods for detecting the smell of explosives include the use of explosive detection dogs trained to detect explosives by smell, and the method of measuring odorants with a mass spectrometer. However, the method of using explosives detection dogs is very costly to train. In addition, the method using a mass spectrometer has problems such as taking a long time for measurement.

On the other hand, a system has been disclosed that analyzes and evaluates odor components by detecting odorants using odorant receptors (OR) expressed in cells (Patent Document 1).

JP 2017-153444 A

However, there are many types of olfactory receptors, and even if a database or the like indicates that a certain olfactory receptor can bind to an odorant, when the olfactory receptor is incorporated into a sensor system, the above-mentioned In some cases, odorants could not be sufficiently detected.

Accordingly, an object of the present invention is to provide a new structural body or a sensor using the same, which can detect an odorant to be detected with high sensitivity.

In order to achieve the above object, the lipid membrane structure for detecting an odorant of the present invention (hereinafter also referred to as "lipid membrane structure") comprises a lipid membrane, an olfactory receptor, and a co-receptor,
The lipid membrane is
having a bag-like structure and containing the olfactory receptor and the co-receptor on the lipid membrane;
the olfactory receptor comprises Or42a receptor and/or Or43b receptor;
The co-receptors include Orco family receptors.

The odorant detection lipid membrane sensor of the present invention (hereinafter also referred to as "lipid membrane sensor") includes a sensor unit,
The sensor unit includes a lipid membrane sensor and an aqueous electrolyte solution,
The lipid membrane sensor includes a lipid membrane, an olfactory receptor, and a co-receptor,
The lipid membrane is
having a bag-like structure and containing the olfactory receptor and the co-receptor on the lipid membrane;
the olfactory receptor comprises Or42a receptor and/or Or43b receptor;
said co-receptors include Orco family receptors;
The aqueous electrolyte solution contains calcium ions,
The sensor unit is arranged in the aqueous electrolyte solution, and when an odorant binds to the olfactory receptor, the odorant enters or exits the aqueous electrolyte solution through the calcium channel between the olfactory receptor and the co-receptor. The calcium ions inside flow in or flow out, and the inflow or flow out of the calcium ions can be measured.

The odorant detection method of the present invention (hereinafter also referred to as "detection method") uses at least one of the odorant-detecting lipid membrane structure of the present invention and the odorant-detecting lipid membrane sensor of the present invention, A detection step of detecting an odorant is included.

A method for producing a lipid membrane structure of the present invention (hereinafter also referred to as a "production method") comprises a step of expressing an olfactory receptor and a co-receptor on a lipid membrane,
The lipid membrane has a bag-like structure,
the olfactory receptor comprises Or42a receptor and/or Or43b receptor;
The co-receptors include Orco family receptors.

According to the present invention, it is possible to provide a new structure or a sensor using the same that can detect an odorant to be detected with high sensitivity.

FIG. 1 is a diagram showing a schematic diagram of a gene-introduced cell in Embodiment 1. FIG. FIG. 2 is a diagram showing an example of an odor molecule. FIG. 3 is a schematic diagram showing the structure of GCaMP. 4 is a graph showing fluorescence intensity in cells after contact with an odorant in Example 1. FIG. 5 is a graph showing fluorescence intensity in cells after contact with an odorant in Example 1. FIG. 6 is a graph showing the current response in cells after contact with an odorant in Example 2. FIG. 7 is a graph showing the current response in cells after contact with an odorant in Example 2. FIG. 8 is a graph showing current responses in cells after contact with an odorant in Example 2. FIG.

As used herein, "lipid membrane" means a membranous body composed of lipids. The lipid membrane may form a planar membrane, or may form a bag-like membrane (vesicle) such as a vesicle (liposome) or a micelle. The lipid membrane may be composed of a single layer of lipid membrane, or may be composed of two or more layers of lipid membrane, preferably a lipid bilayer membrane. When the lipid membrane structure is a cell, the lipid membrane can be called a cell membrane, for example.

Components of the lipid membrane include, for example, cationic lipids; anionic lipids; PEGylated lipids; glycolipids; phospholipid; cholesterol; and the like. The lipid membrane may contain other constituents such as proteins.

As used herein, "lipid membrane structure" means a structure composed of a lipid membrane. The lipid membrane structure may be, for example, a cell-free lipid membrane structure composed of bag-like membranes (vesicles) such as the aforementioned vesicles (liposomes) or micelles, prokaryotes, eukaryotes, or Lipid membrane structures of cell lines such as cells may also be used.

Examples of the cells include animal- or plant-derived cells. Said animal is, for example, a human or a human animal. Examples of the non-human animals include mammals, reptiles, birds, amphibians, vertebrates such as fish; shellfish; insects; Examples of the amphibian cells include Xenopus laevis oocytes. The insect cells include, for example, Sf21 cells (derived from Spodoptera frugiperda ), Sf9 cells (derived from Spodoptera), Tni cells (derived from Trichoplusia ni ), and High Five™ cells (derived from Trichoplusia ni). , S2 cells (Drosophila, Drosophila ) and the like. Examples of the cells include cells isolated from living organisms such as primary cultured cells, cultured cells, and the like. Examples of the mammalian cultured cells include HEK293T cells and Hela cells.

As used herein, "bag-like" or "bag-like structure" means a structure having a closed space (enclosed space) inside. In the present specification, when the "bag-like" or "bag-like structure" is used in combination with the "lipid membrane structure", the "bag-like" or "bag-like structure" means that calcium ions pass through the lipid membrane. , a structure in which movement of calcium ions between the inside and outside of the lipid membrane structure is restricted to such an extent that movement from the inside to the outside and/or movement from the outside to the inside is detectable. The movement of calcium ions can be detected using, for example, a calcium indicator described below.

As used herein, "Or42a receptor" (odorant receptor 42a) is an olfactory receptor derived from Drosophila melanogaster , and forms a heterocomplex with Orco family receptors to detect odorants. Means possible protein.

The Or42a receptor is, for example, a polypeptide consisting of an amino acid sequence (SEQ ID NO: 1) registered in Genbank under accession number: NP_523622. The Or42a receptor is, for example, a protein encoded by a polynucleotide consisting of a base sequence registered in Genbank under accession number: NM_078898.

Amino acid sequence of Or42a receptor (SEQ ID NO: 1)
MDLRRWFPTLYTQSKDSPVRSRDATLYLLRCVFLMGVRKPPAKFFVAYVLWSFALNFCSTFYQPIGFLTGYISHLSEFSPGEFLTSLQVAFNAWSCSTKVLIVWALVKRFDEANNLLDEMDRRITDPGERLQIHRAVSLSNRIFFFFMAVYMVYATNTFLSAIFIGRPPYQNYYPFLDWRSSTLHLALQAGLEYFAMAGACFQDVCVDCYPVNFVLVLRAH MSIFAERLRRLGTYPYESQEQKYERLVQCIQDHKVILRFVDCLRPVISGTIFVQFLVVGLVLGFTLINIVLFANLGSAIAALSFMAAVLLETTPFCILCNYLTEDCYKLADALFQSNWIDEEKRYQKTLMYFLQKLQQPITFMAMNVFPISVGTNISVTKFSFSVFTLVKQMNISEKLAKSEMEE

As used herein, "Or43b receptor" (odorant receptor 43b) is an olfactory receptor derived from Drosophila melanogaster, and forms a heterocomplex with Orco family receptors to detect proteins capable of detecting odorants. means.

Examples of the Or43b receptor include a polypeptide consisting of the amino acid sequence (SEQ ID NO: 2) registered in Genbank under accession number: NP_523656 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 below. The amino acid sequence of SEQ ID NO: 2 below and the amino acid sequence of SEQ ID NO: 3 below differ in the two underlined amino acids, but both are polypeptides that function as Or43b receptors. Further, the Or43b receptor is, for example, a protein encoded by a polynucleotide consisting of a nucleotide sequence registered in Genbank under accession number: NM_078932.

Amino acid sequence of Or43b receptor (SEQ ID NO: 2)
MFGHFKLVYPAPISEPIQSRDSNAYMMETLRNSGLNLKNDFGIGRKIWRVFSFTYNMVILPVSFPINYVIHLAEFPPELLLQSLQLCLNTWCFALKFFTLIVYTHRLELANKHFDELDKYCVKPAEKRKVRDMVATITRLYLTFVVVYVLYATSTLLDGLLHHRVPYNTYYPFINWRVDRTQMYIQSFLEYFTVGYAIYVATATDSYP VIYVAALRTHILLLKDRIIYLGDPSNEG S SDPSYMFKSLVDCIKAHRTMLNFCDAIQPIISGTIFAQFIICGSILGIIMINMVLFADQSTRFGIVIYVMAVLLQTFPLCFYCNAIVDDC K ELAHALFHSAWWVQDKRYQRTVIQFLQKLQQPMTFTAMNIFNINLATNINVAKFAFTVYAIASGMNLDQKLSIKE

Amino acid sequence of Or43b receptor (SEQ ID NO: 3)
MFGHFKLVYPAPISEPIQSRDSNAYMMETLRNSGLNLKNDFGIGRKIWRVFSFTYNMVILPVSFPINYVIHLAEFPPELLLQSLQLCLNTWCFALKFFTLIVYTHRLELANKHFDELDKYCVKPAEKRKVRDMVATITRLYLTFVVVYVLYATSTLLDGLLHHRVPYNTYYPFINWRVDRTQMYIQSFLEYFTVGYAIYVATATDSYP VIYVAALRTHILLLKDRIIYLGDPSNEG T SDPSYMFKSLVDCIKAHRTMLNFCDAIQPIISGTIFAQFIICGSILGIIMINMVLFADQSTRFGIVIYVMAVLLQTFPLCFYCNAIVDDC N ELAHALFHSAWWVQDKRYQRTVIQFLQKLQQPMTFTAMNIFNINLATNINVAKFAFTVYAIASGMNLDQKLSIKE

As used herein, "Orco family receptor" (Orco co-receptor, Or83b) is an olfactory receptor derived from Drosophila melanogaster, and can detect odorants by forming a heterocomplex with the olfactory receptor. protein. Examples of the olfactory receptor include the Or42a receptor and the Or43b receptor.

Examples of the Orco family receptor include a polypeptide consisting of the amino acid sequence (SEQ ID NO: 4) registered in Genbank under accession number: AAT71306 and a polypeptide consisting of the following SEQ ID NO: 5. The amino acid sequence of SEQ ID NO: 4 below and the amino acid sequence of SEQ ID NO: 5 below differ in one amino acid shown underlined, but both are polypeptides that function as Orco family receptors. Further, the Orco family receptor includes, for example, a protein encoded by a polynucleotide consisting of a base sequence registered in Genbank under accession number: AY567998.

Amino acid sequence of Orco family receptor (SEQ ID NO: 4)
MTTSMQPSKYTGLVADLMPNIRAMKYSGLFMHNFTGGSAFMKKVYSSVHLVFLLMQFTFILVNMALNAEEVNELSGNTITTLFFTHCITKFIYLAVNQKNFYRTLNIWNQVNTHPLFAESDARYHSIALAKMRKLFFLVMLT I VASATAWTTITFFGDSVKMVVDHETNSSIPVEIPRLPIKSFYPWNASHGMFYMISFAFQIYYVLFSMIHS NLCDVMFCSWLIFACEQLQHLKGIMKPLMELSASLDTYRPNSAALFRSLSANSKSELIHNEEKDPGTDMDMSGIYSSKADWGAQFRAPSTLQSFGGNGGGGNGLVNGANPNGLTKKQEMMVRSAIKYWVERHKHVVRLVAAIGDTYGAALLLHMLTSTIKLTLLAYQATKINGVNVYAFTVVGYLGYALAQVFHFCIFGNRLIEESSSVMEAAYSCHWY DGSEEAKTFVQIVCQQCQKAMSISGAKFFTVSLDLFASVLGAVVTYFMVLVQLK

Amino acid sequence of Orco family receptor (SEQ ID NO: 5)
MTTSMQPSKYTGLVADLMPNIRAMKYSGLFMHNFTGGSAFMKKVYSSVHLVFLLMQFTFILVNMALNAEEVNELSGNTITTLFFTHCITKFIYLAVNQKNFYRTLNIWNQVNTHPLFAESDARYHSIALAKMRKLFFLVMLT T VASATAWTTITFFGDSVKMVVDHETNSSIPVEIPRLPIKSFYPWNASHGMFYMISFAFQIYYVLFSMIHS NLCDVMFCSWLIFACEQLQHLKGIMKPLMELSASLDTYRPNSAALFRSLSANSKSELIHNEEKDPGTDMDMSGIYSSKADWGAQFRAPSTLQSFGGNGGGGNGLVNGANPNGLTKKQEMMVRSAIKYWVERHKHVVRLVAAIGDTYGAALLLHMLTSTIKLTLLAYQATKINGVNVYAFTVVGYLGYALAQVFHFCIFGNRLIEESSSVMEAAYSCHWY DGSEEAKTFVQIVCQQCQKAMSISGAKFFTVSLDLFASVLGAVVTYFMVLVQLK

Each of said Or42a receptor, said Or43b receptor and/or said Orco family receptor may be a functional equivalent. Said functional equivalent means anything that has the same intended function but differs in structure from the original protein of interest. Examples of functional equivalents of the Or42a receptor, the Or43b receptor, and the Orco family receptor include proteins containing the following polypeptides (1) or (2).
(1) A polypeptide consisting of an amino acid sequence in which one or several amino acids are inserted, substituted, deleted, and/or added to any of the amino acid sequences of SEQ ID NOS: 1-5 (2) SEQ ID NOS: 1-5 A polypeptide consisting of an amino acid sequence having 80% or more identity to any amino acid sequence of

The polypeptide of (1) or (2) forms a heterocomplex with an olfactory receptor that forms a heterocomplex, that is, the Or42a receptor, Or43b receptor, and/or Orco family receptor, It is a polypeptide capable of detecting a substance. When said (1) and (2) are polypeptides derived from the amino acid sequence of SEQ ID NO: 1, said corresponding olfactory receptor is said Orco family receptor. When said (1) and (2) are polypeptides derived from the amino acid sequence of SEQ ID NO: 2 or 3, said corresponding olfactory receptor is said Orco family receptor. When said (1) and (2) are polypeptides derived from the amino acid sequence of SEQ ID NO: 4 or 5, said corresponding olfactory receptor is said Or42a receptor and/or Or43b receptor.

The above "one or several" may be within the range that the polypeptide of (1) above is a functional equivalent. "One or several" of (1) is, for example, 1 to 96, 1 to 80, 1 to 72, 1 to 60, 1 to 48 in the nucleotide sequences of SEQ ID NOs: 1 to 5. , 1-40, 1-24, 1-20, 1-19, 1-16, 1-14, 1-12, 1-9, 1-8, 1-6 , 1 to 3, 1 or 2, 1. In this specification, the numerical range of numbers such as the number of bases or the number of amino acids, for example, discloses all positive integers belonging to the range. That is, for example, the description of "1 to 5" means disclosure of all "1, 2, 3, 4, 5" (the same shall apply hereinafter).

The "identity" may be within the range that the polypeptides of (2) are functional equivalents. The identity of (2) is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more to the amino acid sequences of SEQ ID NOs: 1 to 5. 99% or more. The "identity" can be determined by aligning two base sequences or amino acid sequences (the same applies hereinafter). The alignment can be calculated with default parameters using, for example, BLAST, FASTA, or the like.

As used herein, "odorant" (hereinafter also referred to as "odorant molecule") means a molecule capable of activating an olfactory receptor. In the present specification, when the "odorant" is used in combination with a specific olfactory receptor, the odorant means a molecule capable of activating the specific olfactory receptor. As a specific example, when the odorant is used in combination with the Or42a receptor, the odorant means a substance that enables the movement of calcium ions through calcium channels between the Or42a receptor and the co-receptor. . In addition, when the odorant is used in combination with the Or43b receptor, the odorant means a substance that enables the movement of calcium ions between the Or43b receptor and the co-receptor through the calcium channel.

The odorants include, for example, 2-Butanone, Ethyl butyrate, 2,3-Butanedione, 2-Heptanone, 1-butanol (1 -Butanol), Ethyl trans-2-Butenoate, Isobutyl acetate, Methyl butyrate, cis-2-Hexen-1-ol (Z2-Hexenol), and 2-pentanol (2-Pentanol) and the like. When the olfactory receptor contains the Or42a receptor, the odorants include, for example, 2-butanone, ethyl butyrate, 2,3-butanedione, 2-heptanone, 1-butanol, and methyl butyrate. When the olfactory receptor comprises the Or43b receptor, the odorants are, for example, trans-2-ethyl butyrate, ethyl butyrate, isobutyl acetate, methyl butyrate, cis-2-hexen-1-ol, and 2- Pentanol and the like can be mentioned. The odorant may be an explosive or an odorant emitted from an explosive.

As used herein, "calcium indicator" means a substance whose fluorescence properties change upon binding or dissociation with calcium ions (Ca ²⁺ ). The calcium indicator may be a protein or a compound.

When the calcium indicator is a protein, the calcium indicator may be, for example, a calcium-dependent fluorescent protein or a calcium-dependent fluorescent protein complex.

As used herein, "calcium-dependent fluorescent protein" or "calcium-dependent fluorescent protein complex" means a fluorescent protein or a fluorescent protein complex whose fluorescence properties change depending on the presence or concentration of calcium ions. . The calcium-dependent fluorescent protein and the calcium-dependent fluorescent protein complex form a complex or dissociate depending on, for example, the presence or absence of calcium ions or the concentration of calcium ions. As a result, in the presence of calcium ions, the calcium-dependent fluorescent protein and the calcium-dependent fluorescent protein complex, for example, emit fluorescence by excitation light, or the fluorescence intensity of fluorescence generated by excitation light is enhanced. In the absence of , no fluorescence is emitted by the excitation light, or the fluorescence intensity of the fluorescence generated by the excitation light is attenuated. In the presence of calcium ions, the calcium-dependent fluorescent protein and the calcium-dependent fluorescent protein complex, for example, do not emit fluorescence by excitation light, or the fluorescence intensity of fluorescence generated by excitation light is attenuated. In the absence of the excitation light, fluorescence is emitted or the fluorescence intensity of the fluorescence generated by the excitation light is enhanced. The calcium-dependent fluorescent protein and the calcium-dependent fluorescent protein complex may, for example, increase or decrease the fluorescence intensity of fluorescence generated by excitation light in a calcium ion concentration-dependent manner.

Examples of the calcium ion-dependent fluorescent protein include aequorin. Examples of the calcium-dependent fluorescent protein complex include GCaMP.

As used herein, "GCaMP" is a fusion protein linking circular permutated green fluorescent protein (cpGFP), calmodulin, and the M13 fragment of myosin light chain kinase. Depending, the fluorescence intensity at a given wavelength changes. Specifically, in the presence of calcium ions, calmodulin binds to calcium ions in GCaMP, resulting in a change in the structure of the fusion protein and an increase in fluorescence intensity. The GCaMP is, for example, GCaMP6s (SEQ ID NO: 6, reference 1), G-CaMP (reference 2), G-CaMP1.6 (reference 3), GCaMP2 (reference 4), GCaMP3 (reference 5) , GCaMP4.1 (reference 6), GCaMP5 (reference 7), GCaMP6 (reference 8), GCaMP7 (reference 8), GCaMP8 (reference 8), GCaMP6f (reference 1), GCaMP6m (reference 1 ), jGCaMP7f (reference document 9), jGCaMP7S (reference document 9), jGCaMP7b (reference document 9), jGCaMP7 (reference document 9), and the like. The predetermined wavelength is, for example, a fluorescence wavelength of 495-570 nm. The fluorescence wavelength is, for example, the fluorescence wavelength occurring at an excitation wavelength of 450-495 nm.

Reference 1: Chen, TW. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295-300 (2013).
Reference 2: Nakai, J et al., A high signal-to-noise Ca ²⁺ probe composed of a single green fluorescent protein. Nat Biotechnol 19, 137-141 (2001).
Reference 3: Masamichi Ohkura et al., Genetically Encoded Bright Ca2+ Probe Applicable for Dynamic Ca2 ⁺ Imaging of Dendritic Spines, Analytical Chemistry 2005 77 (18), 5861-5869
Reference 4: Yvonne N. Tallini et al., Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca ²⁺ indicator GCaMP2, PNAS March 21, 2006 103 (12) 4753-4758
Reference 5: Tian, L. et al., Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 6, 875-881 (2009).
Reference 6: Shindo A et al., (2010) Tissue-Tissue Interaction-Triggered Calcium Elevation Is Required for Cell Polarization during Xenopus Gastrulation. PLoS ONE 5(2): e8897
Reference 7: Jasper Akerboom et al. Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging, Journal of Neuroscience 3 October 2012, 32 (40) 13819-13840
Reference 8: Ohkura M et al. (2012) Genetically Encoded Green Fluorescent Ca2+ Indicators with Improved Detectability for Neuronal Ca2+ Signals. PLoS ONE 7(12): e51286.
Reference 9: Dana, H.. et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat Methods 16, 649-657 (2019).

Amino acid sequence of GCaMP6s (SEQ ID NO: 6)
MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKTGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP WPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQ VNYEEFVQMMTAK

Each of said GCaMP6s may be a functional equivalent. Examples of functional equivalents of GCaMP6s include proteins containing the following polypeptides (3) or (4).
(3) A polypeptide consisting of an amino acid sequence in which one or several amino acids are inserted, substituted, deleted, and/or added to the amino acid sequence of SEQ ID NO: 6 (4) For the amino acid sequence of SEQ ID NO: 6, A polypeptide consisting of an amino acid sequence with 80% or more identity

The polypeptide of (3) or (4) is a polypeptide whose fluorescence intensity at a predetermined wavelength changes depending on the concentration of calcium ions.

The above "one or several" may be within the range that the polypeptide of (3) above is a functional equivalent. The "one or several" of (3) is, for example, 1 to 90, 1 to 67, 1 to 45, 1 to 22, 1 to 18, 1 ~13, 1-9, 1-8, 1-6, 1-3, 1 or 2, 1.

The "identity" may be within the range that the polypeptides of (4) are functional equivalents. The identity of (4) with the amino acid sequence of SEQ ID NO: 6 is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.

When the calcium indicator is a compound, examples of the calcium indicator include Quin-2, Fura-2, Indo-1, Fluo-3, Rhod-2 and the like.

The calcium indicator may be placed, for example, inside the lipid membrane structure or outside the lipid membrane structure. preferable. When the calcium indicator is placed inside the lipid membrane structure, the calcium indicator may be placed, for example, on the lipid membrane or inside the bag-like structure of the lipid membrane, that is, the lipid It may be arranged in the internal space of the membrane structure. When the calcium indicator is arranged outside the lipid membrane structure, the calcium indicator is contained, for example, in an electrolyte solution described below.

Changes in the fluorescence properties of the calcium indicator can be detected or measured by conventional methods such as flow cytometry and observation over time using a fluorescence microscope.

As used herein, "electrolyte liquid" means an aqueous solvent containing an electrolyte that ionizes in the aqueous solvent. The electrolyte includes, for example, cations such as potassium ions (K ⁺ ), sodium ions (Na ⁺ ), calcium ions (Ca ²⁺ ), magnesium ions (Mg ²⁺ ), chloride ions (Cl ⁻ ), and phosphate ions. (PO ₄ ^3- ), hydrogen phosphate ion (HPO ₄ ^2- ), dihydrogen phosphate ion (H ₂ PO ₄ ^- ), carbonate ion (CO ₃ ^2- ), hydrogen carbonate ion (HCO ₃ ^- ), etc. It is composed of an anion. The concentration of calcium ions in the electrolyte liquid is, for example, 0.1 to 100 mmol/l, 0.1 to 10 mmol/l, 1 to 10 mmol/l, and 2.5 to 7.5 mmol/l.

The calcium ion concentration of the electrolyte liquid is preferably different from the calcium ion concentration inside the bag-like lipid membrane. Thereby, in the lipid membrane structure of the present disclosure, when the odorant is present, calcium flows from the outside to the inside of the lipid membrane structure or from the inside to the outside through the calcium channel of the olfactory receptor in the lipid membrane structure. A movement of ions occurs, and by detecting this, the presence or absence or concentration of the odorant can be preferably detected. More preferably, the calcium ion concentration of the electrolyte solution is higher than the calcium ion concentration inside the bag-like lipid membrane. Thereby, the lipid membrane structure of the present disclosure can detect, for example, the odorant with higher sensitivity. When the lipid membrane structure is a cell, the calcium ion concentration inside the bag-like lipid membrane is, for example, the cytoplasmic calcium ion concentration. When the presence of the odorant causes calcium ions to move from the outside to the inside of the lipid membrane structure, the concentration of calcium ions inside the lipid membrane structure (C _I ) and the outside of the lipid membrane structure, That is, the calcium ion concentration ratio (C _O /C _I ) to the calcium ion concentration (C _O ) of the electrolyte liquid is, for example, 10 ² to 10 ⁸ , 10 ³ to 10 ⁷ , 10 ³ to 10 ⁶ . , preferably 10 ³ to 10 ⁷ , 10 ³ to 10 ⁶ because the odorant can be detected with higher sensitivity. Further, when calcium ions move from the inside of the lipid membrane structure to the outside when the odorant exists, the concentration of calcium ions inside the lipid membrane structure (C _I ) and the concentration of the lipid membrane structure The calcium ion concentration ratio (C _I /C _O ) to the calcium ion concentration (C _O ) of the external, that is, the electrolyte liquid is, for example, 10 ² to 10 ⁸ , 10 ³ to 10 ⁷ , 10 ³ to 10 ⁶ and preferably 10 ³ to 10 ⁷ , 10 ³ to 10 ⁶ since the odorant can be detected with higher sensitivity.

As used herein, "polynucleotide" means a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and/or modified nucleotides. The polynucleotide may be a single-stranded polynucleotide or a double-stranded polynucleotide.

As used herein, "polypeptide" means a polymer composed of unmodified (naturally occurring), modified, and/or artificial amino acids. Said polypeptide is, for example, a peptide having a length of 10 amino acids or more.

As used herein, "expression vector" or "vector" refers to a recombinant plasmid or virus containing a nucleic acid that is delivered to a host cell in vitro or in vivo .

The terms used herein have the meanings commonly used in the technical field unless otherwise specified.

An example of the present invention will be described below using embodiments and with reference to the drawings. In addition, this invention is not limited to the following embodiment. Also, the descriptions in this specification and each embodiment can be used with each other unless otherwise specified.

(Gene-introduced cells)
In this embodiment, as an example of a lipid membrane structure, the lipid membrane structure is a cell (gene-introduced cell) expressing an olfactory receptor transfected with the gene expression vector, and the lipid membrane is a cell membrane. is an example. FIG. 1 shows a schematic diagram of a gene-introduced cell 1 of this embodiment. As shown in FIG. 1, the transgenic cell 1 of this embodiment expresses an olfactory receptor 20 and a co-receptor 200 on a lipid membrane 10, which is a cell membrane. Olfactory receptors 20 include at least one of Or42a and Or43b receptors. Co-receptors 200 include Orco family receptors. Points other than this are not particularly limited.

In FIG. 1, gene-introduced cell 1 expresses fluorescent protein complex 30 in lipid membrane 10 . The fluorescent protein complex 30 is capable of emitting fluorescence due to structural changes caused by binding with calcium ions (Ca ²⁺ ) 300 . However, it is not limited to this, and the gene-introduced cell 1 may not contain the fluorescent protein complex 30 . As will be described later, the present invention can detect the odorant molecule 100 without including the fluorescent protein complex 30 by using it in combination with a device capable of measuring changes in the concentration of calcium ions.

The transfected cell 1 is not particularly limited, and is, for example, at least one of Sf21 cells and Xenopus laevis oocytes. The Sf21 cells are insect cells derived from Spodoptera frugiperda . However, the gene-introduced cells are not limited to this, and Spodoptera frugiperda -derived Sf9 cells, Trichoplusia ni -derived Tni cells, and High Five cells can also be used.

The gene-introduced cell 1 can be produced, for example, by introducing genes encoding the olfactory receptor 20 and the fluorescent protein complex 30 into the cell and expressing them, as described later.

Olfactory receptors 20 include at least one of Or42a and Or43b receptors. The Or42a receptor and the Or43b receptor are olfactory receptor proteins from Drosophila melanogaster , respectively.

Olfactory receptors 20 can specifically bind to odor molecules 100 . Each of the Or42a receptor and the Or43b receptor can specifically bind to an odorant molecule to be detected.

Specifically, the odor molecules are, for example, 2-butanone, ethyl butyrate, 2,3-butanedione, 2-heptanone ( 2-Heptanone), 1-Butanol, Ethyl trans-2-Butenoate, Isobutyl acetate, Methyl butyrate, cis-2-hexene- 1-ol (Z2-Hexenol), 2-pentanol (2-Pentanol), and the like. When said olfactory receptor comprises said Or42a receptor, said odorant molecules are, for example, 2-butanone, ethyl butyrate, 2,3-butanedione, 2-heptanone, 1-butanol, and methyl butyrate. When the olfactory receptor comprises the Or43b receptor, the odorant molecules are, for example, trans-2-ethyl butyrate, ethyl butyrate, isobutyl acetate, methyl butyrate, cis-2-hexen-1-ol, and 2- Pentanol. Said odorant molecule may be, for example, an odorant molecule emitted from an explosive. The "molecules emitted from explosives" include, for example, substances constituting explosives and impurities generated in the manufacturing process of explosives. The "molecule emitted from the explosive" is, for example, 2-butanone. According to the gene-introduced cell 1 of this embodiment, molecules emitted from explosives can be suitably detected.

Olfactory receptor 20 forms a heterocomplex with Orco family receptor (Olfactory receptor co-receptor; hereinafter also referred to as Orco receptor) 200, for example, as shown in FIG. Form. Orco family receptor 200 is a receptor protein from Drosophila melanogaster .

Fluorescent protein complex 30 is configured to be capable of emitting fluorescence or to change the intensity of fluorescence due to structural changes caused by binding with calcium ions 300 . The fluorescent protein complex 30 is, for example, GCaMP. As shown in FIG. 3, the GCaMP is a protein in which calmodulin, which is a calcium-binding protein, and EGFP, which is a fluorescent protein, bind to each other. In the GCaMP, when calcium ions bind to calmodulin, the structure of EGFP folds and recovers its fluorescence ability, thereby emitting fluorescence. The GCaMP includes multiple variants. Examples of the GCaMP mutant include GCaMP6s.

GCaMP6s can be detected, for example, with an excitation wavelength of 450-495 nm and a fluorescence wavelength of 495-570 nm in a calcium ion-bound state.

In the gene-introduced cell 1 of the present embodiment, when the odorant molecule 100 capable of activating the olfactory receptor 20 is present, the calcium channel formed by the olfactory receptor 20 and the Orco family receptor 200 is opened, and the gene-introduced cell An influx of calcium ions 300 occurs from the outside of 1 to the inside. Further, inside the transfected cell 1, there is a fluorescent protein complex 30 whose fluorescent properties change when bound to calcium ions 300. FIG. Therefore, when the influx of calcium ions 300 from the outside to the inside of the gene-introduced cell 1 occurs, the fluorescent protein complex 30 and the calcium ions 300 bind to each other, and the fluorescent protein 30 emits fluorescence when irradiated with excitation light. Become. Therefore, in the gene-introduced cell 1, the presence or absence of the fluorescence or the intensity change occurs, and the presence and concentration of the odorant molecule 100 can be detected by detecting these changes. On the other hand, in the absence of the odorant molecule 100 capable of activating the olfactory receptor 20, the calcium channel formed by the olfactory receptor 20 and the Orco family receptor 200 remains closed, and the gene-introduced cell 1 is formed. There is no inflow of calcium ions 300 from the outside to the inside. Then, since no new binding occurs between the fluorescent protein complex 30 and the calcium ion 300, the gene-introduced cell 1 does not undergo a change in the presence or absence of fluorescence or a change in intensity. Therefore, in the gene-introduced cell 1, the absence of the odorant molecule 100 can also be detected by detecting the change in the presence or absence of fluorescence or the change in intensity. Therefore, according to the gene-introduced cell 1, the odorant molecule 100 can be detected. In addition, the gene-introduced cell 1 can also be said to be, for example, a gene-introduced cell for detecting the odor molecules. In addition, according to the gene-introduced cell 1, as will be described later, odor molecules emitted from explosives can be detected. The transgenic cell 1 can also be referred to as transgenic cell for detecting explosives, for example.

In this embodiment, an example in which the lipid membrane structure is a cell has been described, but the present invention is not limited to this. Since the lipid membrane structure operates by the mechanism described above, it can be said that a lipid membrane having a bag-like structure having the olfactory receptor and the co-receptor functions similarly.

(Gene introduction vector)
The gene transfer vector of this embodiment is a polynucleotide comprising a base sequence encoding an olfactory receptor and a co-receptor, wherein the olfactory receptor comprises at least one of Or42a receptor and Or43b receptor, Co-receptors include Orco family receptors. The gene introduction vector of the present embodiment is not particularly limited except for these points.

The polynucleotide may, for example, further contain a base sequence encoding a fluorescent protein complex. The fluorescent protein complex is, for example, GCaMP6s.

The olfactory receptor, the co-receptor, and the fluorescent protein complex are as described above.

The olfactory receptor, the co-receptor, and/or the fluorescent protein complex may, for example, be partially or wholly present on the same polynucleotide or on different polynucleotides. good too. In the latter case, the gene transfer vector of the present invention can also be referred to as, for example, a gene transfer vector set.

The gene introduction vector can be constructed, for example, by inserting a polynucleotide encoding a desired protein into a basic vector as follows. The method of inserting the cDNA, the base vector into which the cDNA is inserted, and the like are not particularly limited, and known techniques can be used according to the type of gene to be introduced. The gene introduction vector and the basic vector may contain a replication origin, selection marker, promoter, enhancer, transcription termination sequence (terminator), ribosome binding site, polyadenylation signal and the like.

The cDNA of the Orco receptor (Olfactory receptor co-receptor) gene (accession number: AY567998) derived from Drosophila melanogaster and the Or42a receptor gene (Accession number : AY567998) derived from Drosophila melanogaster Session number: NM_078898) cDNA is inserted into a pIB vector (manufactured by Invitrogen) to construct an Orco-Or42a co-expression vector.

Also, an Orco-Or43b co-expression vector is similarly constructed by using the cDNA of the Or43b receptor gene (accession number: NM — 078932) derived from Drosophila melanogaster instead of the Or42a receptor cDNA.

When the gene introduction vector of this embodiment contains a fluorescent protein complex, a cDNA encoding the fluorescent protein complex is inserted into the base vector to construct a fluorescent protein complex expression vector.

As a specific example, when the fluorescent protein complex is GCaMP6s, a commercially available plasmid (manufactured by Addgene, catalog number: #40753) can be used as the GCaMP6s expression vector, which is the fluorescent protein complex expression vector. .

The gene introduction vector may be, for example, one vector as described above, or a combination of a plurality of vectors (for example, a combination of an olfactory receptor expression vector and a fluorescent protein expression vector).

(Method for producing gene-introduced cells)
This embodiment is an example of producing a gene-introduced cell capable of detecting an odorant by introducing the gene introduction vector into a cell as an example of a method for producing a lipid membrane structure. The method for producing a gene-introduced cell of this embodiment includes the step of expressing the gene-introducing vector in the cell. As a result, in the cell containing the gene transfer vector, the olfactory receptor and co-receptor are expressed on the cell membrane, which is a lipid membrane, and the gene-transferred cell of the embodiment can be produced. The manufacturing method of this embodiment is not particularly limited except for these points.

The production method of the present embodiment may include an introduction step of introducing (also referred to as “transfection”) the gene transfer vector into cells prior to the expression.

The gene transfer vector and the cell are as described above.

The introduction step can be performed, for example, as follows. The method of introducing the gene introduction vector into cells, the culture conditions of the cells, and the like are not particularly limited, and known techniques can be used.

As a first example, the Orco-Or42a co-expression vector and the GCaMP6s expression vector are transfected into Sf21 using a transfection reagent (TransIT (registered trademark)-Insect Transfection Reagent, manufactured by Mirus Bio) according to the attached manual. Introduce into cells. As a result, Sf21 cells co-expressing the Or42a receptor, Orco and GCaMP6s can be obtained. Alternatively, Sf21 cells co-expressing the Or43b receptor, Orco and GCaMP6s can be similarly obtained by using the Orco-Or43b co-expression vector instead of the Orco-Or42a co-expression vector.

As a second example, stage V-VI Xenopus laevis oocytes were treated with a solution containing a collagenase (1.5 mg/ml collagenase, 88 mmol/l NaCl, 1.5 mg/ml collagenase, 88 mmol/l NaCl, 1.5 hours) at 20°C. 0 mmol/l KCl, 2.4 mmol/l _NaHCO3 , 7.5 mmol/l Tris base, 0.8 _mmol /l _MgSO4.7H2O , pH 7.6). mRNAs encoding Or42a receptor and Orco receptor are synthesized using mMESSAGE mMACHINE (manufactured by Ambion), respectively. 25 ng of the synthesized RNA can be introduced into the treated oocytes by microinjection.

The method for producing the gene-introduced cells may further include, for example, a selection step of cloning the transfected cells and selecting a target cell clone from the cloned cell clones.

The selection step can be performed, for example, as follows.

After the introduction, a plurality of types of cell clones are obtained by a limiting dilution method in which the cells are cultured after being diluted so that one well of the plate contains one cell. Thereafter, for example, the multiple types of cell clones are reacted with odorant molecules in a solution, measured with a fluorescence plate reader, and cell clones with higher sensitivity are selected from the multiple types of cell clones.

The production method of the present embodiment may further include the step of culturing the gene-introduced cells and recovering bag-like lipid membrane structures such as exosomes produced from the cells. The recovery can be performed, for example, by recovering the culture medium of the gene-introduced cells. The production method of the present embodiment may further purify the lipid membrane structure from the medium. The purification can be performed, for example, according to an exosome purification method.

In addition, in the production method of the present embodiment, production is performed in an expression system using cells that express the olfactory receptor and the co-receptor in cells, but a cell-free expression system may be used. In this case, a lipid membrane containing the olfactory receptor and the co-receptor is obtained by combining the lipid membrane, a polynucleotide encoding the olfactory receptor and the co-receptor, and a cell-free protein translation system. be able to. As the cell-free expression system, for example, a commercially available kit (ProteoLiposome BD Expression Kit, manufactured by Cell Free Science) can be used.

(cell sensor for odorant detection)
This embodiment is an example of a lipid membrane sensor for detecting an odorant, in which the lipid membrane sensor is the gene-introduced cell (sensor cell). The odorant detection cell sensor of the present embodiment includes a sensor unit. The sensor unit includes sensor cells and an aqueous electrolyte solution. As described above in the gene-introduced cell of the present embodiment, the sensor cell has an olfactory receptor and a co-receptor arranged on the lipid membrane, and the olfactory receptor recognizes at least one of the Or42a receptor and the Or43b receptor. and wherein said co-receptor comprises an Orco family receptor. The aqueous electrolyte solution contains calcium ions. The sensor cells are placed in the aqueous electrolyte solution, and when an odorant binds to the olfactory receptors, calcium channels formed by the olfactory receptors and the co-receptors release the electrolyte into the sensor cells. The calcium ions in the aqueous solution flow in, and the inflow of the calcium ions can be measured. Points other than this are not particularly limited.

The sensor cells may, for example, express fluorescent protein complexes within the lipid membrane. The fluorescent protein complex can emit fluorescence by undergoing a structural change due to binding with calcium ions. Then, the influx of the calcium ions in the aqueous electrolyte solution causes the fluorescent protein complex to emit light. The fluorescent protein complex is, for example, GCaMP6s.

The sensor cells can be, for example, the transgenic cells described above. The olfactory receptor, the co-receptor, and the fluorescent protein complex are as described above.

The aqueous electrolyte solution contains calcium ions. The aqueous electrolyte solution is, for example, an assay buffer (0.1% dimethyl sulfoxide, 140 mmol/l NaCl, 5.6 mmol/l KCl, 4.5 mmol/l CaCl ₂ , 11.26 mmol/l MgCl ₂ , 11.32 mmol/l MgSO ₄ , 9.4 mmol/l D-glucose and 5 mmol/l HEPES, pH 7.2) can be used.

The odorant detection cell sensor is capable of measuring the influx of calcium ions. The inflow of calcium ions may be measured by, for example, fluorescence detection or current measurement. The odorant detection cell sensor can measure, for example, the influx of calcium ions in real time.

The sensor unit may include, for example, the sensor cells and an aqueous electrolyte solution in a container. The sensor cells are, for example, adhered or attached to the container. The container may be one or plural. The container may be capable of culturing the sensor cells. The sensor unit may be, for example, a cell chip.

The odorant-detecting cellular sensor may, for example, further include an odorant-supplying means. The odor substance supplying means is, for example, a tube or the like.

The odorant detection cellular sensor may further include fluorescence detection means, for example. The fluorescence detection means is not particularly limited, and common ones can be used. The fluorescence detection means includes, for example, an image sensor.

When the fluorescent protein complex is, for example, GCaMP6s, the odorant detection cell sensor is, for example, a microplate reader (Infinite F200, TECAN), a microplate reader (Tecan m1000pro, TECAN), or the like. be able to.

For example, the odorant detection cell sensor may further include a current measuring means. The current measuring means measures the inflow of the calcium ions. The fluorescence detection means is not particularly limited, and examples thereof include a whole-cell current measuring device (OC-725C, manufactured by Warner Instruments) using a two-electrode membrane potential clamping method.

The odorant detection cell sensor is, for example, a sensor that detects odor molecules of explosives.

(Odor Substance Detection Method)
This embodiment is an example of using the gene-introduced cell (sensor cell) and the cell sensor for detecting an odorant as an example of an odorant detection method. The odorant detection method of this embodiment includes a detection step of detecting an odorant using at least one of the gene-introduced cell and the odorant detection cell sensor.

As described above, in the transgenic cell and the cell sensor, the binding of the odorant to the olfactory receptor causes an influx of calcium ions into the transgenic cell or cell sensor. Therefore, in the detection step, the odorant can be detected indirectly by detecting changes in the fluorescence properties of the fluorescent protein complex. More specifically, in the detection step, changes in the presence or absence of fluorescence and/or changes in fluorescence intensity of the fluorescent protein complex in the gene-introduced cells and the cell sensor are detected. Then, when the change in the presence or absence of the fluorescence and/or the change in the fluorescence intensity can be detected, it can be evaluated in the detection step that the odorant is present. On the other hand, if the change in the presence or absence of the fluorescence and/or the change in the fluorescence intensity cannot be detected, it can be evaluated that the odorant does not exist in the detection step. Moreover, the detection method of the present embodiment may further analyze the concentration of the odorant from the detection result. In this case, the detection method of the present embodiment can analyze the concentration of the odorant using, for example, a calibration curve showing the correlation between the change in fluorescence intensity and the amount or concentration of the odorant. For the calibration curve, for example, a plurality of known amounts or concentrations of odorants are prepared, and each of these is brought into contact with the gene-introduced cells and/or the cell sensor, and the amount of change in the fluorescence intensity is detected. Then, the calibration curve can be prepared, for example, using the amount or concentration and the amount of change in fluorescence intensity.

The detection step includes, for example, an odorant supply step of supplying an odorant to the gene-introduced cells, an irradiation step of irradiating excitation light, and a detection step of detecting fluorescence. The supply step, the irradiation step, and the detection step can refer to the descriptions of Examples described later. In the irradiation step, the excitation light may have a wavelength of, for example, 450 to 495 nm. In the detection step, the fluorescence wavelength to be detected can be, for example, 495 to 570 nm.

The odorant detection method of the present embodiment may, for example, further include a determination step of determining whether or not an odorant is contained based on the detected fluorescence. Specifically, the determination step includes, for example, the fluorescence intensity when the odorant and the transfected cells are brought into contact, and the fluorescence intensity when the odorant-free control substance and the transfected cells are brought into contact. The determination can be made based on the amount of change in the fluorescence intensity by comparing the fluorescence intensity.

Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercially available reagents were used according to their protocol unless otherwise indicated.

[Example 1]
Gene-introduced cells, which are the lipid membrane structures of the present invention, were prepared, and it was confirmed that odorants could be detected.

(Preparation of expression vector)
The cDNA of the Orco receptor (Olfactory receptor co-receptor) gene (accession number: AY567998) derived from Drosophila melanogaster and the Or42a receptor gene (Accession number : AY567998) derived from Drosophila melanogaster Session number: NM_078898) cDNA was inserted into a pIB vector (manufactured by Invitrogen) to construct an Orco-Or42a co-expression vector. In addition, a commercially available plasmid (manufactured by Addgene, catalog number # 40753) was used as the GCaMP6s expression vector.

An Orco-Or43b co-expression vector was similarly constructed using the cDNA of the Or43b receptor gene (accession number: NM — 078932) derived from Drosophila melanogaster instead of the Or42a receptor cDNA.

(Preparation of gene-introduced cells)
The Orco-Or42a co-expression vector and the GCaMP6s expression vector were introduced into Sf21 cells using a transfection reagent (TransIT (registered trademark)-Insect Transfection Reagent, manufactured by Mirus Bio) according to the attached manual. This yielded Sf21 cells co-expressing the Or42a receptor, Orco and GCaMP6s. Further, Sf21 cells co-expressing the Or43b receptor, Orco, and GCaMP6s were similarly obtained using the Orco-Or43b co-expression vector instead of the Orco-Or42a co-expression vector.

After the introduction, cloning was performed by a limiting dilution method in which one well of the plate contains one cell and then cultured to obtain a cell clone of each of the cells.

The cell clones were cultured with 10 μg/ml gentamicin (ThermoFisher), 10 μg/ml blasticidin (ThermoFisher), 100 μg/ml zeocin (ThermoFisher), 10% fetal bovine serum (GE It was carried out using Grace's insect medium (manufactured by Gibco) containing (manufactured by Healthcare).

(Preparation of odorant-containing solution)
As odorant molecules that bind to the Or42a receptor and the Or43b receptor, 2-butanone, ethyl butyrate, 2,3-butanedione, and 2 shown in FIG. - 2-Heptanone, 1-Butanol, Ethyl trans-2-Butenoate, Isobutyl acetate, Methyl butyrate, cis-2 -Hexen-1-ol (Z2-Hexenol), and 2-Pentanol (2-Pentanol) were used. The odor molecules were added to assay buffer (0.1% dimethyl sulfoxide, 140 mmol/l NaCl, 5.6 mmol/l KCl, 4.5 mmol/l CaCl ₂ , 11.26 mmol/l MgCl ₂ , 11.32 mmol/l MgSO ₄ , 9.4 mmol/l D-glucose and 5 mmol/l HEPES, pH 7.2). The concentration of calcium ions in the odorant-containing solution is about 10 ⁴ to 10 ⁵ times the concentration of calcium ions in the cytoplasm of the gene-introduced cells.

(Measurement of fluorescence)
0.1×10 ⁶ cells of the cells in the logarithmic growth phase were seeded in a 96-well plate and cultured at room temperature for 1 hour. Then, the medium was removed, 100 μl of the assay buffer containing the odorant was added, and the fluorescence intensity (FI) was measured using a microplate reader (Infinite F200, TECAN) under the following measurement conditions, and ΔFI (( (measured value - measured value of density)/measured value of 0 density) x 100 (%)) was calculated. The "measured value at 0 concentration" indicates the measured value when the same experiment was performed using the assay buffer containing no odor molecules.

(Measurement condition)
Excitation wavelength: 485 nm (bandwidth: 20 nm)
Fluorescence (detection) wavelength: 540 nm (bandwidth: 25 nm)
Laser intensity (Gain): 90

The results are shown in FIGS. 4 and 5. FIG. 4 and 5 are graphs showing fluorescence intensity in the cells after contact with the odorants, respectively. In FIGS. 4 and 5, the horizontal axis indicates the odorant, and in each case, 0.3 mmol/l of the odorant was used on the left, and 3 mmol/l of the odorant was used on the right. The vertical axis indicates the fluorescence intensity (ΔFI (%)).

FIG. 4 shows that 0.3 mmol/l of Ethyl butyrate, 2,3-Butanedion, 2-Heptanone, and 1-Butanol and 3 mmol/l of 2 as the odorants were added to the Or42a receptor-expressing cells. -Butanone, Ethyl butyrate, 2,3-Butanedion, 2-Heptanone, 1-Butanol, and 3 mmol/l of 2-Butanone are shown. As shown in FIG. 4, the fluorescence intensity increased when 0.3 mmol/l of the odorant was used. Moreover, when 3 mmol/l of the odorant was used, the fluorescence intensity increased more than when 0.3 mmol/l of the odorant was used.

FIG. 5 shows the results of contacting the Or43b receptor-expressing cells with Ethyl trans-2-Butenoate, Isobutyl acetate, Methyl butyrate, Z2-Hexenol, 2-Pentanol, and Ethyl butyrate as the odorants. . As shown in FIG. 5, the fluorescence intensity increased when 0.3 mmol/l and 3 mmol/l of the odorant were used.

As described above, it was confirmed that the gene-introduced cells of the present invention could be produced and odorants could be detected.

[Example 2]
Gene-introduced cells of the present invention were produced under conditions different from those in Example 1, and it was confirmed that odorants could be detected. It was also confirmed that the gene-introduced cells of the present invention can detect odorants in a concentration-dependent manner.

(Preparation of gene-introduced cells)
Stage V-VI Xenopus laevis oocytes were treated at 20° C. for 1.5 hours with a solution containing a collagenase (1.5 mg/ml collagenase, 88 mmol/l NaCl, 1.0 mmol/l KCl, 2 .4 mmol/l _NaHCO3 , 7.5 mmol/l Tris base, _0.8 mmol/l _MgSO4.7H2O , pH 7.6). mRNAs encoding Or42a receptor and Orco receptor were each synthesized using mMESSAGE mMACHINE (registered trademark, Ambion). 25 ng of the synthesized RNA was introduced into the treated oocytes by microinjection. The introduced oocytes were treated at 20° C. with a Barth solution (88 mmol/l NaCl, 1.0 mmol/l KCl, 2.4 mmol/l NaHCO ₃ , 7.5 mmol/l Tris base, 0.8 mmol/l l _MgSO4.7H2O , 0.33 mmol/ _l Ca( _NO3 )2.4H2O, 0.41 mmol _/ l CaCl2.2H2O, 10 units _/ ml penicillin _G , 10 mg/ml _streptomycin sulfate, pH 7 .6) and cultured for 2-3 days.

(Preparation of odorant-containing solution)
Methyl butyrate, Ethyl butyrate, and 2-butanone shown in FIG. 2 were used as odor molecules that bind to the Or42a receptor. As control odor molecules, Isobutyl acetate and Ethyl trans-2-butenoate shown in FIG. 2 were used. After dissolving each odorant in DMSO (dimethyl sulfoxide), assay buffer (96 mmol/l NaCl, 2.0 mmol/l KCl, 2.5 mmol/l HEPES, 2.5 mmol/l MES, 1.8 mmol/l _CaCl2.2H2O , 1.6 mmol/l _MgCl2.6H2O , pH _7.5 ) by diluting with 300 μmol/l odorant- _containing solution was made. Also, as a control, a 0.1% DMSO solution was prepared. The concentration of calcium ions in the odorant-containing solution is about 10 ⁴ to 10 ⁵ times the concentration of calcium ions in the cytoplasm of the gene-introduced cells.

(Measurement of membrane current)
Whole-cell current measurement was performed on the oocytes by a two-electrode membrane voltage clamp method (OC-725C, manufactured by Warner Instruments). Data acquisition and analysis used Digidata 1322A and pCLAMP software (Axon Instruments). The holding potential was set to −80 mV and inward currents were measured. The cells were then stimulated by adding 40 μl of the odorant-containing solution to the oocyte chamber.

The results are shown in FIGS. 6(A) and (B) and FIG. FIG. 6 is a graph showing the time course of the current response in the cell upon contact with the odorant, (A) showing the results of control, Methyl butyrate, Isobutyl acetate, and Ethyl trans-2-butenoate; (B) shows the results for Ethyl butyrate and 2-Butanone. In FIG. 6, the horizontal axis indicates time (seconds) and the vertical axis indicates current (nA). As shown in FIG. 6, as a result of stimulation with Methyl butyrate, Ethyl butyrate, and 2-butanone, current responses were observed as indicated by arrows in the figure. On the other hand, when Isobutyl acetate and Ethyl trans-2-butenoate were used as the odorant, no clear current response was observed.

Figure 7 is a graph showing the current response in the cell upon contact with the odorant. In FIG. 7, the horizontal axis indicates the odorant, and the vertical axis indicates the current (maximum value after stimulation with each odorant) (nA). In addition, the value of the said electric current was made into the average value of the measurement value of a total of 12 times. As shown in FIG. 7, as a result of stimulation with Methyl butyrate, Ethyl butyrate, and 2-Butanone, currents of 30 nA, 22.7 nA, and 15.1 nA were measured, respectively. The value of the electric current increased compared with the case of using. On the other hand, when Ethyl trans-2-Butenoate was used as the odorant, no significant increase in current value was observed as compared with the case of using the 0.1% DMSO solution. Further, when isobutyl acetate was used as the odorant, the current value tended to increase only slightly.

Next, using Methyl butyrate, Ethyl butyrate, and 2-Butanone as the odorant molecules, the final concentrations in the odorant-containing solution were adjusted to 10, 30, 100, 300, 1000, and 3000 μmol/l, respectively, Membrane currents were measured in the same manner, except that DMSO was adjusted to a final concentration of 1%.

The results are shown in FIG. Figure 8 is a graph showing the current response in the cell upon contact with the odorant. In FIG. 8, the horizontal axis indicates concentration (μmol/l) and the vertical axis indicates current (nA). As shown in FIG. 8, the current value increased as the concentration of the odorants increased in all cases of using Methyl butyrate, Ethyl butyrate, and 2-butanone. In particular, Methyl Butyrate and Ethyl Butyrate significantly increased the current value at 300, 1000 and 3000 μmol/l compared to the case of using the 1% DMSO solution (p<0. 01).

As described above, the gene-introduced cells of the present invention were produced under conditions different from those in Example 1, and it was confirmed that odorants could be detected. It was also confirmed that the gene-introduced cells of the present invention can detect odorants in a concentration-dependent manner.

Although the present invention has been described with reference to the embodiments, the present invention is not limited to the above embodiments. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

<Appendix>
Some or all of the above-described embodiments can be described as the following notes, but are not limited to the following.
<Lipid membrane structure for detecting odorants>
(Appendix 1)
comprising a lipid membrane, an olfactory receptor and a co-receptor,
The lipid membrane is
having a bag-like structure and containing the olfactory receptor and the co-receptor on the lipid membrane;
the olfactory receptor comprises Or42a receptor and/or Or43b receptor;
said co-receptors include Orco family receptors;
A lipid membrane structure for detecting odorants.
(Appendix 2)
Supplementary note 1, wherein when the olfactory receptor binds to an odorant, a signal associated with the influx of calcium ions (Ca ²⁺ ) into the pouch-like structure or outflow from the pouch-like structure can be detected. The described lipid membrane structure.
(Appendix 3)
containing a calcium indicator,
The lipid membrane structure according to appendix 1 or 2, wherein the calcium indicator is arranged inside the bag-like structure of the lipid membrane.
(Appendix 4)
The lipid membrane structure according to Appendix 3, wherein the calcium indicator is a calcium-dependent fluorescent protein complex.
(Appendix 5)
The lipid membrane structure according to Appendix 4, wherein the calcium-dependent fluorescent protein complex is GCaMP6s.
(Appendix 6)
6. The lipid membrane structure according to any one of Appendices 1 to 5, wherein the lipid membrane is a lipid bilayer membrane.
(Appendix 7)
the lipid membrane structure is a cell,
7. The lipid membrane structure according to any one of Appendices 1 to 6, wherein the lipid membrane is a cell membrane.
(Appendix 8)
The lipid membrane structure according to appendix 7, wherein the cells are at least one of Sf21 cells and Xenopus laevis oocytes.
(Appendix 9)
8. The lipid membrane structure according to any one of Appendices 1 to 7, wherein the lipid membrane structure is a vesicle.
(Appendix 10)
the olfactory receptor is the Or42a receptor,
10. The lipid membrane structure according to any one of Appendices 1 to 9, wherein the odorants are 2-butanone, ethyl butyrate, 2,3-butanedione, 2-heptanone, 1-butanol, and methyl butyrate.
(Appendix 11)
the olfactory receptor is the Or43b receptor,
11. The odorant according to any one of appendices 1 to 10, wherein the odorant is trans-ethyl 2-butenoate, ethyl butyrate, isobutyl acetate, methyl butyrate, cis-2-hexen-1-ol, and 2-pentanol. Lipid membrane structure.
<Vector for gene introduction>
(Appendix 12)
comprising a polynucleotide encoding an olfactory receptor and a co-receptor;
the olfactory receptor comprises Or42a receptor and/or Or43b receptor;
said co-receptors include Orco family receptors;
A vector for gene transfer.
(Appendix 13)
13. The gene transfer vector according to Appendix 12, wherein the polynucleotide comprises a polynucleotide encoding a calcium-dependent fluorescent protein complex.
(Appendix 14)
14. The gene transfer vector according to Appendix 13, wherein the calcium-dependent fluorescent protein complex is GCaMP6s.
<Odorous substance detection lipid membrane sensor>
(Appendix 15)
including a sensor unit,
The sensor unit includes a lipid membrane sensor and an aqueous electrolyte solution,
The lipid membrane sensor includes a lipid membrane, an olfactory receptor, and a co-receptor,
The lipid membrane is
having a bag-like structure and containing the olfactory receptor and the co-receptor on the lipid membrane;
the olfactory receptor comprises Or42a receptor and/or Or43b receptor;
said co-receptors include Orco family receptors;
The aqueous electrolyte solution contains calcium ions,
The sensor unit is arranged in the aqueous electrolyte solution, and when an odorant binds to the olfactory receptor, the odorant enters or exits the aqueous electrolyte solution through the calcium channel between the olfactory receptor and the co-receptor. The calcium ions in the inflow or outflow are configured to be able to measure the inflow or outflow of the calcium ions.
Lipid membrane sensor for odorant detection.
(Appendix 16)
Attachment 15, wherein when the olfactory receptor binds to an odorant, a signal associated with the influx of calcium ions (Ca ²⁺ ) into the pouch-like structure or outflow from the pouch-like structure can be detected. The described lipid membrane sensor.
(Appendix 17)
containing a calcium indicator,
17. The lipid membrane sensor according to appendix 15 or 16, wherein the calcium indicator is arranged inside the bag-like structure of the lipid membrane.
(Appendix 18)
18. The lipid membrane sensor according to appendix 17, wherein the calcium indicator is a calcium-dependent fluorescent protein complex.
(Appendix 19)
19. The lipid membrane sensor according to appendix 18, wherein the calcium-dependent fluorescent protein complex is GCaMP6s.
(Appendix 20)
20. The lipid membrane sensor according to any one of appendices 15 to 19, wherein the lipid membrane is a lipid bilayer membrane.
(Appendix 21)
the lipid membrane structure is a cell,
21. The lipid membrane sensor according to any one of appendices 15 to 20, wherein the lipid membrane is a cell membrane.
(Appendix 22)
22. The lipid membrane sensor according to appendix 21, wherein the cells are at least one of Sf21 cells and Xenopus laevis oocytes.
(Appendix 23)
22. The lipid membrane sensor according to any one of Appendices 15 to 21, wherein the lipid membrane structure is a vesicle.
(Appendix 24)
the olfactory receptor is the Or42a receptor,
24. The lipid membrane sensor according to any one of Appendices 15 to 23, wherein the odorants are 2-butanone, ethyl butyrate, 2,3-butanedione, 2-heptanone, 1-butanol, and methyl butyrate.
(Appendix 25)
the olfactory receptor is the Or43b receptor,
25. according to any one of appendices 15 to 24, wherein said odorant is trans-ethyl 2-butenoate, ethyl butyrate, isobutyl acetate, methyl butyrate, cis-2-hexen-1-ol, and 2-pentanol Lipid membrane sensor.
<Detection method>
(Appendix 26)
A detection step of detecting an odorant using at least one of the odorant-detecting lipid membrane structure according to any one of Appendices 1 to 11 and the odorant-detecting lipid membrane sensor according to any one of Appendices 15 to 25. A method for detecting an odorant, comprising:
(Appendix 27)
27. The detection method according to attachment 26, wherein in the detection step, the inflow or outflow of the calcium ions into or out of the lipid membrane sensor caused by the binding of the odorant and the olfactory receptor is detected.
(Appendix 28)
The odorant-detecting lipid membrane structure or the odorant-detecting lipid membrane sensor contains a calcium indicator,
In the detection step, the inflow or outflow of the calcium ions into or out of the lipid membrane sensor caused by the binding of the odorant and the olfactory receptor is detected by detecting a change in the fluorescence properties of the calcium indicator. , the detection method according to appendix 26 or 27.
(Appendix 29)
29. The detection method according to any one of attachments 26 to 28, comprising an analysis step of analyzing at least one of the presence or absence and concentration of the odorant from the detection result in the detection step.
<Method for producing lipid membrane structure>
(Appendix 30)
A step of expressing an olfactory receptor and a co-receptor on a lipid membrane,
The lipid membrane has a bag-like structure,
the olfactory receptor comprises Or42a receptor and/or Or43b receptor;
A method for producing a lipid membrane structure, wherein the co-receptor includes an Orco family receptor.
(Appendix 31)
the lipid membrane is the cell membrane of a cell,
Supplementary note 30. The production method according to Supplementary note 30, comprising the step of introducing the gene transfer vector according to any one of Supplementary notes 12 to 14 into the cell.
(Appendix 32)
32. The production method according to Appendix 31, comprising a recovery step of culturing the obtained cells and recovering the bag-like lipid membrane structure.

According to the present invention, it is possible to provide a new structure capable of detecting an odorant to be detected with high sensitivity. According to the present invention, the odorants can also be measured in real time and at low cost by detecting changes in fluorescence properties. Therefore, the present invention is very useful in fields such as detection of explosives.

Claims (10)

comprising a lipid membrane, an olfactory receptor and a co-receptor,
The lipid membrane is
having a bag-like structure and containing the olfactory receptor and the co-receptor on the lipid membrane;
the olfactory receptor comprises Or42a receptor and/or Or43b receptor;
said co-receptors include Orco family receptors;
A lipid membrane structure for detecting odorants. 2. When the olfactory receptor binds to an odorant, a signal associated with the influx of calcium ions (Ca ²⁺ ) into the pouch-like structure or outflow from the pouch-like structure can be detected. Lipid membrane structure according to. further comprising a calcium-dependent fluorescent protein complex,
3. The lipid membrane structure according to claim 1, wherein said calcium-dependent fluorescent protein complex is GCaMP6s. the lipid membrane structure is a cell,
The lipid membrane structure according to any one of claims 1 to 3, wherein the lipid membrane is a cell membrane. The lipid membrane structure according to claim 4, wherein the cells are at least one of Sf21 cells and Xenopus laevis oocytes. the olfactory receptor is the Or42a receptor,
The lipid membrane structure according to any one of claims 1 to 5, wherein the odorants are 2-butanone, ethyl butyrate, 2,3-butanedione, 2-heptanone, 1-butanol, and methyl butyrate. the olfactory receptor is the Or43b receptor,
7. Any one of claims 1 to 6, wherein the odorant is trans-ethyl 2-butenoate, ethyl butyrate, isobutyl acetate, methyl butyrate, cis-2-hexen-1-ol, and 2-pentanol. Lipid membrane structure according to. including a sensor unit,
The sensor unit includes a lipid membrane sensor and an aqueous electrolyte solution,
The lipid membrane sensor includes a lipid membrane, an olfactory receptor, and a co-receptor,
The lipid membrane is
having a bag-like structure and containing the olfactory receptor and the co-receptor on the lipid membrane;
the olfactory receptor comprises Or42a receptor and/or Or43b receptor;
said co-receptors include Orco family receptors;
The aqueous electrolyte solution contains calcium ions,
The sensor unit is arranged in the aqueous electrolyte solution, and when an odorant binds to the olfactory receptor, the odorant enters or exits the aqueous electrolyte solution through the calcium channel between the olfactory receptor and the co-receptor. The calcium ions in the inflow or outflow are configured to be able to measure the inflow or outflow of the calcium ions.
Lipid membrane sensor for odorant detection. A detection step of detecting an odorant using at least one of the odorant-detecting lipid membrane structure according to any one of claims 1 to 7 and the odorant-detecting lipid membrane sensor according to claim 8. A method for detecting an odorant, comprising: A step of expressing an olfactory receptor and a co-receptor on a lipid membrane,
The lipid membrane has a bag-like structure,
the olfactory receptor comprises Or42a receptor and/or Or43b receptor;
A method for producing a lipid membrane structure, wherein the co-receptor includes an Orco family receptor.

Images