WO2021045233A1

WO2021045233A1 - Odor detection kit, odor detection kit manufacturing method, and odor detection method

Info

Publication number: WO2021045233A1
Application number: PCT/JP2020/033831
Authority: WO
Inventors: 秀文光野; 章吾荒木; 駿佑藤林; 大悟照月; 健志櫻井; 哲志山口; 久美子小熊; 亮平神崎; 佐和子二木; 侑司祐川
Original assignee: 国立大学法人東京大学
Priority date: 2019-09-06
Filing date: 2020-09-07
Publication date: 2021-03-11
Also published as: JPWO2021045233A1

Abstract

An odor detection kit for a fluorometer, said odor detection kit comprising: a substrate 10; and odor detection cells 20 that are positioned in a single layer on the substrate 10, have olfactory receptors, and express a fluorescent protein, the fluorescence intensity thereof changing in accordance with the concentration of odor molecules.

Description

Smell detection kit, manufacturing method of odor detection kit, and odor detection method

The present invention relates to a detection technique, a method for manufacturing an odor detection kit, and a method for detecting an odor.

Water is obtained from a water source, treated at a water purification plant, and then supplied as tap water to homes, stores, and business establishments. Alternatively, food factories and the like acquire water from their own water sources and use the water to produce beverages and food. Examples of water sources include dam lakes, natural lakes, rivers, and groundwater. When the temperature of the water source rises, floating algae and actinomycetes are generated, and the water obtained from the water source may have a musty odor. The musty odor of water may not disappear even after purified water. Therefore, it is desirable to quantify the musty odor of water before supplying it to consumers.

The degree of musty odor of water can be accurately quantified by gas chromatography-mass spectrometry (GC / MS). However, since it takes time to carry out GC / MS, even if the GC / MS detects a musty odor exceeding the specified value when water purification and supply are continuously carried out, it is a sample source. Water may already be supplied to consumers. In addition, since the device for performing GC / MS is difficult to move, the device for performing GC / MS cannot be used to evaluate the musty odor of water immediately after being obtained from an outdoor water source. Further, although the inspector can perform a sensory evaluation of the musty odor of water, it takes time to train the inspector, and the result of the sensory evaluation can vary depending on the inspector.

Therefore, a simple odor inspection method different from GC / MS has been proposed (see, for example, Patent Documents 1 to 3). However, these methods may not be able to inspect odors stably and accurately.

Japanese Patent No. 5127783 Japanese Patent No. 6474945 Special Table 2002-518305

There is a demand for a technology that can easily and stably detect not only the musty odor of water but also the odor of various inspection targets. Therefore, one of the objects of the present invention is to provide an odor detection kit capable of easily and stably detecting an odor, a method for producing an odor detection kit, and a method for detecting an odor.

According to the aspect of the present invention, an odor detection cell having a substrate and a single layer arranged on the substrate, and expressing a fluorescent protein whose fluorescence intensity changes according to the concentration of odor molecules. And, an odor detection kit for a fluorometer is provided.

In the above odor detection kit, the odor detection cells may be fixed to the substrate via a cell membrane modifier.

The above odor detection kit may further include a tube capable of fixing the substrate inside.

In the above odor detection kit, the odor detection cells may express the olfactory receptor by the transgene.

In the above odor detection kit, the odor detection cells may express the olfactory receptor of an insect.

In the above odor detection kit, the olfactory receptor may be an ion channel type receptor.

In the above odor detection kit, the fluorescent protein may change the fluorescence intensity according to the ion concentration.

In the above odor detection kit, the olfactory receptor may be selected from BmOR1, BmOR3, DmOr13a, DmOr56a, DmOr82a, DmOr49b, DmOr85b and PxOR1.

In the above odor detection kit, the odor detection cell may be an insect cell.

In the above odor detection kit, the insect cell may be a cell derived from ga.

In the above odor detection kit, insect cells may be selected from Sf21, Sf9, High Five, and Tni-derived cells.

In the above odor detection kit, the insect cells may be cells derived from Drosophila.

In the above odor detection kit, the insect cells may be Drosophila S2 cells.

In the above odor detection kit, the substrate may be transparent.

Further, according to the aspect of the present invention, an odor detection cell having an olfactory receptor and expressing a fluorescent protein whose fluorescence intensity changes according to the concentration of an odor molecule is prepared, and a substrate is prepared. A method for producing an odor detection kit for a fluorometer is provided, which comprises arranging odor detection cells in a single layer on a substrate.

In the above method for manufacturing an odor detection kit, odor detection cells may be fixed to a substrate via a cell membrane modifier.

The above-mentioned method for manufacturing an odor detection kit may further include fixing a substrate on which odor detection cells are arranged in a tube.

The method for producing the above-mentioned odor detection kit is as follows: (a) a part of cells are selected from a group of cells having an olfactory receptor and expressing a fluorescent protein, and (b) the selected cells are proliferated. c) Performing multiple steps (a) to (c) to confirm the responsiveness of the proliferated cells to the odorant, and odorizing the proliferated cells whose responsiveness to the odorant is equal to or higher than the standard value. It may further include selection as detection cells.

The method for producing the above-mentioned odor detection kit is as follows: (a) a part of cells are selected from a group of cells having an olfactory receptor and expressing a fluorescent protein, and (b) the selected cells are proliferated. c) Perform multiple steps (a) to (c) to confirm the responsiveness of the proliferated cells to the odorant, and select the proliferated cells with the highest responsiveness to the odorant as odor detection cells. And may further include.

The above-mentioned method for producing an odor detection kit may include expressing a second olfactory receptor in cells expressing the first olfactory receptor. The first olfactory receptor and the second olfactory receptor may be different. The first olfactory receptor and the second olfactory receptor may react with different odor molecules.

In the above method for producing an odor detection kit, some of the selected cells may be a single cell.

Further, according to the aspect of the present invention, an odor detecting cell having an olfactory receptor arranged in a single layer on a substrate and expressing a fluorescent protein whose fluorescence intensity changes according to the concentration of an odor molecule. , An odor detection method is provided that includes contacting the fluid to be tested for odor and measuring the fluorescence emitted by the odor detecting cells.

In the above odor detection method, the odor detection cells may be fixed to the substrate via a cell membrane modifier.

In the above odor detection method, the substrate may be fixed in the tube, and the above odor detection method may further include putting a fluid in the tube.

In the above odor detection method, the fluid may be a liquid.

According to the present invention, it is possible to provide an odor detection kit capable of easily and stably detecting an odor, a method for producing an odor detection kit, and a method for detecting an odor.

It is a schematic diagram of the odor detection kit for the fluorometer according to the embodiment. It is a schematic diagram which shows the method which expresses the olfactory receptor which concerns on embodiment in an odor detection cell. It is a schematic diagram which shows the method of fixing the odor detection cell to the substrate which concerns on embodiment. It is a schematic diagram which shows the method of homogenizing the odor detection cell which concerns on embodiment. It is a schematic diagram which shows the method of expressing a plurality of olfactory receptors according to an embodiment in an odor detection cell. It is a schematic diagram which shows the method of expressing a plurality of olfactory receptors according to an embodiment in an odor detection cell. It is a schematic diagram which shows the response of the odor detection cell to the odor molecule which concerns on embodiment. It is a photograph and the graph which show the concentration-dependent response of the odor detection cell to the odor molecule which concerns on Example. It is a graph which shows the specific response of the odor detection cell to the odor molecule which concerns on Example. It is a graph which shows the time change of the fluorescence emitted by the suspension of the odor detection cell which concerns on Example. It is a graph which shows the response to the odor molecule of the homogenized odor detection cell and the non-homogenized odor detection cell which concerns on Example. It is a graph which shows the response to the odor molecule of the odor detection cell which was subcultured for a long time which concerns on an Example. It is a graph which shows the response to the odor molecule of the odor detection cell which was subcultured for a long time after the frozen storage of 3 and a half months which concerns on Example. It is a graph which shows the response to the odor molecule of the odor detection cell which was subcultured for a long time after freezing storage for 6 and a half months which concerns on Example. It is a graph which shows the measurement result using the odor detection kit which concerns on Example. It is a graph which shows the relationship between the geosmin concentration which concerns on Example, and the change rate of the detected fluorescence intensity. It is a graph which shows the odor molecule specificity of the odor detection kit which concerns on Example. It is a graph which shows the time comparison until the odor detection kit and the cell suspension which concerns on an Example become stable. It is a graph which shows the responsiveness comparison to the odor molecule of the odor detection kit and the cell suspension which concerns on Example. It is a graph which shows the variation comparison of the responsiveness to the odor molecule of the odor detection kit and the cell suspension which concerns on Example. It is a graph which shows the measurement result in the outdoors using the odor detection kit which concerns on Example. It is a graph which shows the time change of the fluorescence emitted by the odor detection cell which concerns on Example. It is a graph which shows the time change of the fluorescence emitted by the odor detection cell which concerns on Example. It is a graph which shows the time change of the fluorescence emitted by the odor detection cell which concerns on Example. It is a graph which shows the relationship between the change of fluorescence emitted by the odor detection cell which concerns on Example, and the concentration of an odor substance. It is a graph which shows the time change of the fluorescence emitted by the odor detection cell which concerns on Example. It is a graph which shows the relationship between the change of fluorescence emitted by the odor detection cell which concerns on Example, and the concentration of an odor substance. It is a graph which shows the relationship between the change of fluorescence emitted by the odor detection cell which concerns on Example, and an odor substance.

An embodiment of the present invention will be described below. In the description of the drawings below, the same or similar parts are represented by the same or similar reference numerals. However, the drawings are schematic. Therefore, the specific dimensions and the like should be determined in light of the following explanations. In addition, it goes without saying that the drawings include parts having different dimensional relationships and ratios from each other.

As shown in FIG. 1, the odor detection kit for a fluorometer according to the embodiment has a substrate 10 and an olfactory receptor arranged in a single layer on the substrate 10, depending on the concentration of odor molecules. It comprises an odor detecting cell 20 expressing a fluorescent protein whose fluorescence intensity changes.

The substrate 10 is transparent, for example, and is made of glass, resin, or the like.

The odor detection cell 20 expresses an olfactory receptor on the cell membrane. In the odor detection cell 20, the olfactory receptor may be naturally expressed or may be expressed by a transgene. The olfactory receptor may be an insect olfactory receptor.

The odor detection cell 20 may be an insect cell. Insect cells may be cells derived from moths such as Spodoptera frugiperda and Cabbage looper (Trichoplusia ni). Examples of cells derived from Spodoptera frugiperda include Sf21 and Sf9. Sf21 cells are derived from ovarian cells. Sf21 cells can divide infinitely and establish a stable expression line that permanently expresses the introduced gene. In addition, Sf21 cells can survive in a wide temperature range of 18 ° C to 40 ° C and do not require carbon dioxide to adjust the pH of the culture medium. Originally, Sf21 cells do not have an olfactory receptor, but it is possible to express an olfactory receptor by introducing an olfactory receptor gene. Sf9 cells are clones of Sf21. Examples of cells derived from cabbage looper include High Five and Tni. Tni-derived cells are derived from ovarian cells.

Alternatively, the insect cell may be a cell derived from Drosophila. Examples of Drosophila-derived cells include Drosophila S2 cells.

The olfactory receptor may be a G protein-coupled receptor or an ion channel receptor. The ion channel type receptor has a site that interacts with a ligand that is an odor molecule and a site through which ions flow. When the ion channel type receptor of the odor detecting cell 20 binds to the ligand, cations such as sodium ion and calcium ion flow into the odor detecting cell 20. In the odor detection cell 20, the influx of ions can occur within a few tens of milliseconds from the binding of the ligand. The amount of inflow to ion Many, for binding one ligand, the amount of ions flowing into the cell is said to be 10 ^7.

In general, certain types of olfactory receptors have specificity for certain odor molecules. In the odor detection cell 20, only one type of olfactory receptor corresponding to one type of odor molecule may be expressed, or a plurality of types of olfactory receptors corresponding to a plurality of types of odor molecules may be expressed. In addition, the amount of olfactory receptor to be expressed may be adjusted.

Examples of olfactory receptors are DmOr56a, which is a receptor for Drosophila and is a receptor for geosmine, which has a musty odor, and DmOr82a, which is a receptor for geranyl acetate, which is a receptor for Drosophila and has an aromatic or fruity odor. DmOr49b, a receptor for Drosophila and a receptor for 2-methylphenol (o-cresol) having a human sweat odor, and 1-octen-3-, a receptor for Drosophila and a musty odor. DmOr13a, which is a receptor for ol, BmOR1, which is a receptor for Bombycol, which is a sex pheromone of Kaikoga, BmOR3, which is a receptor for Bombykal, which is a subcomponent of the sex pheromone of Kaikoga, and general Examples include, but are not limited to, the odor receptor DmOr85b and the sex pheromone receptor of Konaga, PxOR1. In addition, "geosmin" is also called "geosmin".

When the olfactory receptor is expressed in the odor detection cell 20 by genetic engineering, for example, as shown in FIG. 2, the gene encoding the olfactory receptor is incorporated into the vector, and the constructed vector is transfected into the host cell. The gene encoding the olfactory receptor can be isolated, for example, by extracting mRNA from the olfactory organ of an insect and synthesizing the cDNA. From the isolated cDNA, it is possible to amplify a part of the gene encoding the olfactory receptor by the PCR method using PCR primers.

A part of the gene encoding the olfactory receptor can also be obtained by incorporating the synthesized double-stranded cDNA into an appropriate vector and transforming Escherichia coli or the like with the vector to prepare a cDNA library. it can. The cDNA can be incorporated into a vector by a usual method using a restriction enzyme and a ligase, for example, by cutting the obtained cDNA with a restriction enzyme, inserting it into the restriction enzyme site of the vector DNA, and ligating it to the vector.

Fluorescent protein is expressed in the odor detection cell 20. As described above, in the odor detecting cell 20, when an odor molecule binds to the ion channel type olfactory receptor, ions flow in the odor detecting cell 20. Therefore, by introducing a gene that expresses a fluorescent protein whose fluorescence intensity changes according to ions into the odor detecting cell 20, it is confirmed whether or not the odor detecting cell 20 detects the odor molecule from the change in the fluorescence intensity. It is possible to do. Examples of fluorescent proteins include GCaMP3, GCaMP6s and aequorin, which fluoresce by reacting with calcium ions.

When the concentration of odor molecules is high, the concentration of ions flowing into the odor detection cells 20 is also high, and many fluorescent proteins fluoresce. Therefore, the fluorescence intensity emitted by the odor detecting cells 20 becomes stronger. When the concentration of odor molecules is low, the concentration of ions flowing into the odor detection cells 20 is also low, and a small amount of fluorescent protein fluoresces. Therefore, the fluorescence intensity emitted by the odor detecting cells 20 is weakened. Therefore, it is possible to evaluate the concentration of odor molecules based on the fluorescence intensity.

In addition, the interaction between odor molecules and odor molecule receptors is weak, and the same odor molecule interacts with the same odor molecule receptor many times, or the same odor molecule interacts with multiple odor molecule receptors of the same cell. Or, the same odor molecule can be thought of as interacting with odor molecule receptors in multiple different cells. Therefore, it is considered that the odor molecules can be detected with high sensitivity because the ion inflow into the cell is repeatedly generated at least with the number of odor molecules.

The surface of the substrate 10 shown in FIG. 1 may be coated with a protein such as collagen or bovine serum albumin (BSA). The odor detection cells 20 may be immobilized on the substrate 10 via a protein that covers the surface of the substrate 10. Alternatively, the odor detection cells 20 may be immobilized on the substrate via a cell membrane modifier. Alternatively, as shown in FIG. 3, the odor detecting cell 20 may be fixed on the substrate 10 via a cell membrane modifier and a protein such as collagen or BSA that covers the surface of the substrate 10.

The cell membrane modifier has a cell membrane binding group that binds to the cell membrane of the odor detection cell 20. Lipids can be used as the cell membrane binding group. For example, as the cell membrane binding group, an oleyl group, a diorail group, a cholesterol group, and a tetraethylene glycol group can be used. In addition, the cell membrane modifier has a substrate-binding group that binds to the surface of the substrate 10 or the protein that covers the substrate 10. An N-hydroxysuccinimide (NHS) group can be used as the substrate-binding group that binds to the protein that covers the substrate 10. The cell membrane modifier may have polyethylene glycol (PEG) that enhances water solubility between the cell membrane binding group and the substrate binding group. In order to keep an appropriate distance between the substrate 10 and the odor detecting cells 20, the number of repetitions of ethylene oxide units of PEG may be appropriately set.

Examples of the cell membrane modifier include oleyl-O-poly (ethylene glycol) succinyl-N-hydroxy-succinimidyl ester (oleyl-PEG-NHS) represented by the following chemical formula (1). Note that n represents a natural number.

As shown in FIG. 1, the odor detection kit for the fluorometer according to the embodiment further includes a tube 30 to which the substrate 10 can be fixed inside. For example, by making the inner diameter of the tube 30 and the width of the substrate 10 substantially the same, it is possible to suppress the substrate 10 inserted in the tube 30 from moving in the tube 30.

When the odor detecting cell 20 is a non-adhering cell, the odor detecting cell 20 may not adhere to the surface of the substrate 10 even if the odor detecting cell 20 is brought into contact with the surface of the substrate 10. Alternatively, even if the odor detecting cell 20 is an adherent cell, the adhesive force to the substrate 10 is weak and the odor detecting cell 20 may be easily peeled off from the substrate 10. On the other hand, if a cell membrane modifier is used, even if the odor detection cells 20 are non-adhesive cells or adherent cells with weak adhesion, the odor detection cells 20 are fixed to the surface of the substrate 10 with a single layer. It is possible.

Next, a method of manufacturing an odor detection kit for a fluorometer according to the embodiment will be described. In the method for producing an odor detection kit for a fluorometer according to the embodiment, odor detection cells 20 having an olfactory receptor and expressing a fluorescent protein whose fluorescence intensity changes according to the concentration of odor molecules are prepared. This includes preparing the substrate 10 and arranging the odor detection cells 20 in a single layer on the substrate 10.

The odor detection cell 20 (a) selected some cells from the group of cells having an olfactory receptor and expressing a fluorescent protein, (b) proliferated the selected cells, and (c) proliferated. A plurality of steps (a) to (c) for confirming the responsiveness of the cell to the odorant may be carried out, and the responsiveness to the odorant may be selected from the proliferated cells having a reference value or more. The cell selected in step (a) may be a single cell (single cell).

Alternatively, the odor detection cell 20 may (a) select some cells from the group of cells having an olfactory receptor and expressing a fluorescent protein, (b) proliferate the selected cells, and (c) A plurality of steps (a) to (c) for confirming the responsiveness of the proliferated cells to the odorant may be carried out, and the proliferated cells having the highest responsiveness to the odorant may be selected. The cell selected in step (a) may be a single cell (single cell).

Specifically, as shown in FIG. 4, a single cell or a small number of cells are selected by repeating dilution of a cell lineage having an olfactory receptor and expressing a fluorescent protein, and the selected cells are selected. A cell line may be established by culturing and proliferating. By carrying out a plurality of the steps, a plurality of cell lines are established. Among the plurality of established cell lines, a cell line whose responsiveness to an odorant is equal to or higher than a predetermined reference value may be used as the odor detection cell 20. Alternatively, among the plurality of established cell lines, the cell line having the highest responsiveness to the odorant may be used as the odor detection cell 20.

As shown in FIGS. 5 and 6, another olfactory receptor may be expressed in the cells expressing the established olfactory receptor. That is, the second olfactory receptor may be further expressed in the cells expressing the first olfactory receptor established by the above method or the like. For example, by incorporating a gene encoding a second olfactory receptor into a vector and transfecting the constructed vector into cells expressing the first olfactory receptor, the first olfactory receptor and the second olfactory receptor are used. It is possible to establish cells expressing the olfactory receptor of. A plurality of different olfactory receptors may be further expressed in cells expressing the first olfactory receptor. FIG. 5 shows that a vector containing a gene for Or-X, which is a second olfactory receptor, and a vector containing an antibiotic resistance gene are introduced into cells expressing Or56a as the first olfactory receptor. An example is shown. FIG. 6 shows an example of introducing a vector containing both the gene of Or-X, which is the second olfactory receptor, and the antibiotic resistance gene into cells expressing Or56a as the first olfactory receptor. There is.

When the odor detection cells 20 are arranged in a single layer on the substrate 10 shown in FIG. 1, the odor detection cells 20 may be fixed to the substrate 10 via a cell membrane modifier as described above. For example, the substrate 10 is coated with a protein, and a solution containing a cell membrane modifier is dropped onto the protein covering the substrate 10 to react the protein covering the substrate 10 with the substrate binding group of the cell membrane modifier to cause the substrate 10 to react. The cell membrane modifier is bound to the protein that covers the protein. Then, a solution containing the odor detecting cell 20 is dropped onto the substrate 10 to react the cell membrane-binding group of the cell membrane modifier bound to the substrate 10 with the odor detecting cell 20 to modify the cell membrane on the substrate 10. The odor detection cells 20 are fixed via the agent.

After that, the substrate 10 on which the odor detection cells 20 are arranged is fixed in the tube 30.

Next, the odor detection method according to the embodiment will be described. The odor detection method according to the embodiment is an odor detection cell 20 having an olfactory receptor arranged in a single layer on a substrate 10 and expressing a fluorescent protein whose fluorescence intensity changes according to the concentration of odor molecules. Includes contacting the fluid 40 to be tested for odor and measuring the fluorescence emitted by the odor detecting cells 20.

When the substrate 10 is fixed in the tube 30, the tube 30 may contain a buffer solution or a solution containing a substance necessary for the survival of the odor detecting cells 20 as appropriate. By putting the fluid 40 to be tested for odor in the tube 30, the fluid 40 to be tested for odor may be brought into contact with the odor detecting cells 20. The fluid 40 to be tested for odor is a liquid such as water.

For example, after the fluid 40 to be tested for odor is put into the tube 30, the tube 30 is placed on a fluorometer and the odor detection cells 20 are irradiated with excitation light. As shown in FIG. 7, the odor detecting cell 20 irradiated with the excitation light emits intense fluorescence corresponding to the concentration of the odor molecule contained in the fluid 40 to be inspected for odor. When the fluorescent protein is GCAMP6s, the wavelength of the excitation light is, for example, 495 nm, and the wavelength of fluorescence is 510 nm to 580 nm.

The odor of the fluid 40 to be inspected for odor is evaluated based on the intensity of fluorescence emitted by the odor detection cells 20. For example, the odor of the fluid 40 may be quantified based on the relationship between the intensity of fluorescence acquired in advance and the concentration of odor molecules and the intensity of odor. Alternatively, when the intensity of fluorescence emitted by the odor detecting cell 20 is equal to or higher than a preset reference value, it may be evaluated that the odor of the fluid 40 to be tested for odor is equal to or higher than the reference value.

According to the odor detection method according to the present embodiment, the odor of the fluid 40 to be tested for odor is easily evaluated by bringing the fluid 40 to be tested for odor into contact with the odor detection cells 20 fixed to the substrate 10 in advance. It is possible to do.

Further, in the odor detection method according to the present embodiment, for example, when a portable desktop fluorometer is used as a fluorometer, the odor of the fluid 40 to be inspected for odor can be detected in a short time not only in the laboratory but also in any place. It is possible to evaluate with. For example, when the fluid 40 to be inspected for odor is water and the odor to be inspected is a musty odor, it is possible to collect water from an outdoor water source and inspect the musty odor of the water on the spot. ..

(Example)
(Example 1: Preparation of cells expressing DmOr56a)
From the start codon to the stop codon of the codon DmOrco gene derived from the antennal cDNA of Drosophila melanogaster was amplified with a primer containing the following gene-specific sequence to obtain the DmOrco gene (full length of ORF). .. The obtained DmOrco gene was inserted into a multicloning site of a pIB vector (manufactured by Invitrogen) to construct a pIB-DmOrco vector.
DmOrco:
Forward: 5'-TTCGAATTTAAAGCTGCCGCCATGACAACCTCTATGCAACC-3'
Reverse: 5'-TTACCTTCGAACCGCTTACTTAAGCTGAACAAGAACCATGAAG-3'

Gene amplification by PCR uses forward and reverse primers at a concentration of 100 pmol / L, PrimeSTAR HS DNA polymerase (Takara Bio Co., Ltd. R010A), the reaction buffer attached to the polymerase, and dNTP, and attaches to the polymerase. I followed the protocol of. The PCR temperature conditions are a step of 94 ° C. for 2 minutes, then a step of repeating a temperature cycle of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds, 72 ° C. for 2 minutes for 30 cycles, and then at 72 ° C. for 10 minutes. It was a step of.

Similarly, the start codon to stop codon of the receptor DmOr56a gene derived from the tactile cDNA of Drosophila melanogaster is amplified with a primer containing the following gene-specific sequence to obtain the DmOr56a gene (full length of ORF). Obtained. The obtained DmOr56a gene was inserted into the multi-cloning site of the pIB vector to construct the pIB-DmOr56a vector.
DmOr56a:
Forward: 5'-CAGTGTGGTGGAATTGCCGCCATGTTTAAAGTTAAGGATCTGTTGC-3'
Reverse: 5'-GCCCTCTAGACTCGACTAATACAAGTGGGAGCTAC-3'

Next, the protein expression cassette of the pIB-DmOrco vector (the portion linked to the OpIE2 promoter (P (OpIE2)), DmOrco, and OpIE2 polyA addition signal (OpIE2pA)) was amplified and amplified to the Pci1 site of the pIB-DmOr56a vector. The protein expression cassette was inserted to construct the olfactory receptor expression vector pIB-DmOr56a-DmOrco.

In addition, a calcium sensitive protein (GCaMP6s) expression vector was constructed. The GCaMP6s gene was obtained from Dr. Douglas Kim (Janelia Farm Research Campus, Howard Hughes Medical Institute) via Addgene. From the start codon to the stop codon of the GCaMP6s gene was amplified with a primer containing the following gene-specific sequence to obtain the GCaMP6s gene (full length of ORF). The obtained GCaMP6s gene was inserted into a multicloning site of a pIZ vector (manufactured by Invitrogen) to construct a pIZ-GCaMP6s vector.
GCaMP6s:
Forward: 5'-TTCGAATTTAAAGCTGCCGCCATGGGTTCTCATCATCATCATC-3'
Reverse: 5'-TTACCTTCGAACCGCTCACTTCGCTGTCATCATTTGTAC-3'

The constructed olfactory receptor expression vector and calcium-sensitive protein expression vector were introduced into Sf21 cells by the lipofection method (TransIT-Insect; registered trademark of Mirus) according to the attached manual of TransIT-Insect. As a result, Sf21 cells expressing DmOr56a receptor and co-receptor DmOrco, and calciu-sensitive protein (GCaMP6s) were obtained. The DmOr56a receptor reacts with musty odor molecules.

(Example 2: Concentration dependence of response of DmOr56a expressing cells)
The assay buffer (140 mmol / L NaCl) was added to 0 mol / L, 30 nmol / L, 100 nmol / L, 300 nmol / L, 1 μmol / L, 3 μmol / L, 10 μmol / L, and 30 μmol / L of geosmine, which is the causative agent of musty odor. Serial dilution with 5.6 mmol / L KCl, 4.5 mmol / L CaCl ₂ , 11.26 _{mmol / L MgCl 2} , 10 mmol / L HEPES, 9.4 mmol / L D-glucose, pH 7.2) did. The assay buffer for response measurement and the odor solution were all prepared to contain a final concentration of 0.1% DMSO. Next, each of the diluents was contacted with Sf21 cells expressing DmOr56a receptor and co-receptor DmOrco, and GCaMP6s. As a result, as shown in FIG. 8, Sf21 cells expressing DmOr56a receptor and co-receptor DmOrco, and GCaMP6s emitted intense fluorescence according to the concentration of geosmin.

(Example 3: Smell molecule specificity of response of DmOr56a expressing cells)
A solution containing linalol at a concentration of 10 μmol / L, a solution containing decanal at a concentration of 10 μmol / L, a solution containing octanal at a concentration of 10 μmol / L, and a solution containing citral at a concentration of 10 μmol / L, and a concentration of 10 μmol / L. A solution containing geosmin was prepared in. Linalool is an odor molecule that produces lily of the valley, lavender, and bergamot-like scents. Decanal is an odor molecule that produces a citrus scent. Octanal is an odor molecule that gives rise to the scent of fruits. Citral is an odor molecule that gives the scent of lemon.

The solution containing each odor molecule was contacted with Sf21 cells expressing DmOr56a receptor and co-receptor DmOrco, and GCaMP6s. As a result, as shown in FIG. 9, Sf21 cells expressing DmOr56a receptor and co-receptor DmOrco, and GCaMP6s reacted specifically with geosmin to fluoresce.

(Example 4: Detection of odor molecules using a suspension of DmOr56a expressing cells)
Sf21 cells expressing DmOr56a receptor and co-receptor DmOrco, and GCaMP6s were adherently cultured in a T-25 flask. The cells were then stripped from the T-25 flask with a scraper (IWAKI; 9020-250) and the cell suspension was transferred to a 50 mL tube. After centrifuging the tube at 500 xg, the supernatant was removed from the tube.

To the tube, 20 mL of assay buffer was added and the cell pellet was suspended in assay buffer. The tube was then centrifuged at 500 xg. After centrifugation, the supernatant was removed from the tube ^{, assay buffer was added to the tube at 1 × 10 7} cells / mL, and the cells were suspended to obtain a cell suspension.

Place 180 μL of cell suspension in a 0.5 mL tube (Promega; E4941) and place the tube on a fluorometer (Quantus, Fluorometer, registered trademark) to determine the base fluorescence intensity as shown in FIG. It was measured. The base fluorescence intensity was stabilized by repeating pipetting and fluorescence measurement for 5 seconds until the fluorescence intensity with a fluctuation of ± 2% or less could be obtained 10 times in succession. After the base fluorescence intensity was stabilized, 20 μL of 10 μmol / L geosmin was added dropwise to the cell suspension in the tube. After the dropping, the fluorescence intensity was measured every 20 seconds. As a result, it was confirmed that the cells fluoresce in response to geosmin. However, it has been shown that it takes time for the base fluorescence intensity to stabilize if the cells are not immobilized.

(Example 5: Establishment of homogeneous odor detection cell line)
Grace's Insec Medium, Supermented (Gibco; 11605-094), US Insect Cell Selected FBS (GE Healthcare; SH30070.03) with a final concentration of 10%, and 3 types of antibiotics (final concentration of 10 μg / mL Gentamicin) Prepare a subculture medium by adding Reagent Solution (Gibco; 15710-064), Blasticidin S HCl (Gibco; A11139-03) with a final concentration of 10 μg / mL, and Zeocin (Invitrogen; R25001) with a final concentration of 100 μg / mL. did. Using the subculture medium, Sf21 cells expressing DmOr56a receptor and co-receptor DmOrco, and GCaMP6s were subcultured in a flask (FALCON; 353082). The volume of the cell suspension at the time of passage was 6 mL. When the cells became confluent, 6 mL of supernatant was collected from the flask and placed in a 15 mL tube (TPP; 91015). A 15 mL tube was centrifuged at 400 xg at 4 ° C. for 3 minutes using a micro high speed centrifuge.

After centrifugation, the supernatant was sterilized using a 10 mL syringe (TOP; 01007) and a 0.45 μm filter (CORNING; 431220). A 10 mL condition medium was prepared by mixing the sterilized supernatant with an equal volume of new passage medium containing antibiotics (Blastidin S HCl with a final concentration of 10 μg / mL, Zeocin with a final concentration of 100 μg / mL). ..

The cells adhered to the bottom of the flask from which the supernatant was collected were peeled off and suspended in 1 mL of a new medium, and the cell suspension was collected in a 1.5 mL tube (AXYGEN; MCT-150-C). A cell suspension containing 40 cells was extracted, added to the above-mentioned condition medium, and pipetted well. The entire volume of the condition medium to which the cells were added was transferred to a reservoir (BMBio; BM-0850-1). Further, using an 8-channel pipette (HTL; HT5123), 100 μL of the condition medium in which the cells were added to a 96-well plate (IWAKI; 3860-096) was added dropwise, and then the cells were cultured at 27 ° C. After the cells adhered to the wells, the wells were observed under an inverted microscope to confirm the wells in which only single cells (single cells) were present in the condition medium.

The cells in the wells where single cells were confirmed at the time of seeding were continuously cultured until they became about 80% to about 90% confluent. The cells were then scaled up in the order of a 24-well plate (IWAKI; 3820-024), a 35 mm dish (CORNING; 353801), and a T-25 flask. For culturing in a 24-well plate, a 35 mm dish, and a T-25 flask, the amount of medium was adjusted so that the liquid volumes were 500 μL, 2.5 mL, and 5 mL, respectively, and the cells were cultured at 27 ° C. The responsiveness of the cells scaled up to the T-25 flask was investigated by calcium imaging, and the cell line showing good responsiveness was obtained as a homogeneous odor detection cell line.

(Example 6: Reactivity of homogeneous odor detection cell line)
Sf21 cells expressing DmOr56a receptor and co-receptor DmOrco, and GCaMP6s, which are not homogenized, and Sf21 cells expressing DmOr56a receptor and co-receptor DmOrco, and GCaMP6s. The fluorescence response of the homogenized Sf21 cells to the odorant was measured by the following procedure.

After the cells were seeded and adhered to a cover glass (CS-12R: Warner Instruments, LLC) having a diameter of 12 mm, the cover glass was inserted into an open bath chamber (RC-48LP: Warner Instruments, LLC) for a circular cover glass. .. For perfusion of the solution in the chamber and addition of odor molecules, a silicon tube with an inner diameter of 1 mm and an outer diameter of 3 mm connected to a peristaltic tube pump (MP-2010: Tokyo Rikakai Co. Ltd.) was placed in the chamber by a self-made holder. It was installed at the inlet and outlet respectively. The cells were stimulated under the perfusion conditions with a flow rate of about 1.5 mL / min and a volume of liquid in the chamber of about 200 μL, and an odor stimulation time of 10 μmol / L with geosmin was set to 15 seconds.

The fluorescence response of the cells in the chamber was measured using a microscope. As a microscope, an upright fluorescence microscope (BX51WI: Olympus) equipped with a 20x water immersion objective lens (UMPlanFI 20x / 0.50 W: Olympus) was used. An EM-CCD camera (DU-897E: Andor Technology PLC) was used to measure the change in fluorescence intensity of cells. As for the operation of the camera, an image of 512 × 512 pixels was acquired using AndoriQ (Andor Technology). A fluorescent filter set for GFP (U-MGFPHQ: Olympus) was used as the filter of the microscope. A 100 W halogen lamp (TH4-100, Olympus) was used as the light source, and the exposure time during fluorescence observation was set to 500 milliseconds. Shooting was done every second.

FIG. 11 shows a graph showing the relationship between the rate of change in fluorescence intensity before and after giving an odor molecule and the ratio of the number of cells. When a non-homogenized cell line was used, 67.7% of the cells had a fluorescence reaction of 5% or more. In addition, many cells had a low rate of change in fluorescence when an odor molecule was given, and the average rate of change was 26.7%. On the other hand, when the homogenized cell line was used, 92.0% of the cells had a fluorescence reaction. In addition, many cells had a high rate of change in fluorescence when an odor molecule was given, and the average rate of change was 45.9%. Therefore, it was shown that odor molecules can be detected with high sensitivity when a homogenized cell line is used.

(Example 7: Long-term stability of odor detection cells)
A homogenized cell line having a fluorescence change rate of 36.1% at the time of odor stimulation immediately after cloning was passaged for a long period of time, and the change in fluorescence change rate at the time of odor stimulation was examined. As a result, as shown in FIG. 12, the rate of change in fluorescence with respect to the odor stimulus equal to or higher than that immediately after cloning was shown for three and a half months after cloning. In addition, the homogenized cell line was frozen and stored for 3 and a half months immediately after cloning, and after thawing, the cell line was subcultured for a long period of time, and the change in the fluorescence change rate at the time of odor stimulation was examined. As a result, as shown in FIG. 13, the fluorescence change rate was equal to or higher than that immediately after cloning for 2 months after thawing. In addition, the homogenized cell line was stored frozen for 6 and a half months immediately after cloning, and after thawing, the cell line was subcultured for a long period of time, and the change in the fluorescence change rate at the time of odor stimulation was examined. As a result, as shown in FIG. 14, for one and a half months after thawing, the fluorescence change rate was equal to or higher than that immediately after cloning with respect to the odor stimulus.

(Example 8: Preparation of odor detection kit)
A 76 mm × 26 mm slide glass (MATSUNAMI, S9441) was cut into 4 mm × 26 mm with a glass cutting diamond cutter (TRUSCO, 419-9847). Approximately 100 μL of collagen solution was added dropwise to the cut glass, and the slide glass was allowed to stand in a clean bench for 30 minutes. The collagen solution used was Cellmatic Type IC (Nitta Gelatin Co., Ltd., 63100771) diluted 10-fold with HCl (Fujifilm Wako Pure Chemical Industries, Ltd., pH = 3).

After 30 minutes, the excess collagen solution was removed from the slide glass, an appropriate amount of a 100 μmol / L cell membrane modifier (oleyl-PEG-NHS) solution was added dropwise to the slide glass, and the mixture was allowed to stand at 37 ° C. for 30 minutes. The cell membrane modifier solution was prepared by diluting 0.4 mg of SUNBRIGHT (NOF, OE-040CS) and 10 mL of ultra-dehydrated DMSO 100-fold with D-PBS (Fuji Film Wako Pure Chemical Industries, Ltd., 045-29795). After 30 minutes, the excess cell membrane modifier solution was removed from the slide glass and allowed to stand for 20 minutes.

After 20 minutes, ^{a suspension of about 1 × 10 7} homogenized odor-detecting cells (solvent was assay buffer) was added dropwise to a slide glass surface treated with a cell membrane modifier, and the mixture was allowed to stand for 10 minutes. Then, the odor detection kit according to the example was prepared by fixing a slide glass in which odor detection cells were fixed with a single layer in a 0.5 mL tube filled with 400 μL of assay buffer containing 0.1% DMSO.

(Example 9: Smell measurement using a kit and a fluorometer)
Immediately after preparing the odor detection kit according to the example, in order to confirm that the base fluorescence intensity becomes stable, the odor detection kit according to the example is mounted on a fluorometer (Quantus, Fluorometer, registered trademark). It was installed and waited until 10 points with a range of change rate of fluorescence intensity within ± 2% could be measured. Next, 200 μL of 10 μmol / L geosmin was added to 400 μL of the assay buffer in the tube of the odor detection kit according to the example, and the solution in the tube was pipetted for 10 seconds. Then, the rate of change in fluorescence intensity was measured every 20 seconds for 140 seconds. As a result, as shown in FIG. 15, the fluorescence response to geosmin was measured. When the same measurement was repeated using the odor detection kits according to a plurality of examples, the fluorescence response to geosmin was stably measured in each of them. If the slide glass was not fixed in the tube, the base fluorescence intensity was not stable, and the fluorescence response to geosmin could not be measured stably.

(Example 10: Detection of low-concentration odor molecules using a kit and a fluorometer)
Geosmin was added to the odor detection kit according to the examples so that the final concentrations were 0 mol / L, 10 pmol / L, 100 pmol / L, 1 nmol / L, 10 nmol / L, 100 nmol / L, 1 μmol / L, and 10 μmol / L, respectively. Then, the rate of change in fluorescence intensity was measured with a fluorometer (Quantus, Fluorometer, registered trademark). The results are shown in FIG. When the geosmin concentration is 0 mol / L, the rate of change in fluorescence intensity is 1.5 ± 0.4%, and when the concentration of geosmin is 10 pmol / L, the rate of change in fluorescence intensity is 2.7 ± 0.5%. Yes, the rate of change in fluorescence intensity when the concentration of geosmin is 100 pmol / L is 5.5 ± 1.0%, and the rate of change in fluorescence intensity when the concentration of geosmin is 1 nmol / L is 8.1 ± 0.8. %, The rate of change in fluorescence intensity when the concentration of geosmin is 10 nmol / L is 12.0 ± 1.0%, and the rate of change in fluorescence intensity when the concentration of geosmin is 100 nmol / L is 21.3 ± 2. When the concentration of geosmin is 1.0% and the concentration of geosmin is 1 μmol / L, the rate of change in fluorescence intensity is 35.0 ± 5.3%, and when the concentration of geosmin is 10 μmol / L, the rate of change in fluorescence intensity is 29.4. It was ± 4.3%, and the fluorescence intensity increased depending on the concentration of geosmin.

As a result of testing the measured rate of change in fluorescence intensity by the significance level obtained by correcting the p-value calculated by Welch's t-test by the Holm method, geosmin of 100 pmol / L or more was compared with geosmin of 0 mol / L. A significant difference was observed. Therefore, it was shown that the odor molecule can be detected with the same sensitivity as GC / MS by using the odor detection kit and the fluorometer according to the examples. For example, the concentration of geosmin in the dam lake, which is the source of tap water in Japan, was 33.5 nmol / L or less. Therefore, it was shown that the musty odor of water collected from the dam lake can be detected by using the odor detection kit and the fluorometer according to the examples. In addition, the lower limit of human sensory evaluation of the geosmin solution prepared as described above was about 10 nmol / L. Therefore, it was shown that a low-concentration mold odor that cannot be detected by a human sensory evaluation can be detected by using the odor detection kit and the fluorometer according to the examples.

(Example 11: Specific detection of odor molecules using a kit and a fluorometer)
Multiple types of musty odors such as geosmin and 2-methylisoborneol (2-MIB) are generated in the dam lake water. Therefore, in order to evaluate the mold odor selectivity of the odor detection kit according to the prepared example, geosmin 10 μmol / L, 2-MIB 10 μmol / L, and an assay buffer (control) containing neither mold odor were used. It was added dropwise to the odor detection kit according to the example, and the rate of change in fluorescence intensity was measured with a fluorometer (Quantus, Fluorometer, registered trademark).

As a result, as shown in FIG. 17, the rate of change in fluorescence intensity when the control was dropped was 0.1 ± 0.6%, and the rate of change in fluorescence intensity when 2-MIB was dropped was −0.3 ±. The rate of change in fluorescence intensity was 1.1%, and the rate of change in fluorescence intensity when geosmin was added dropwise was 13.7 ± 3.2%. As a result of testing the measured rate of change in fluorescence intensity by the significance level obtained by correcting the p-value calculated by Welch's t-test by the Holm method, a significant difference was observed at 10 μmol / L geosmin compared with the control. No significant difference was observed in 2-MIB of 10 μmol / L. Therefore, it was shown that the odor detection kit according to the prepared example specifically detects geosmin.

(Example 12: Comparison of cell suspension and kit stability)
The suspension of odor detection cells to which no odor molecule was added was placed on a fluorometer (Quantus, Fluorometer, registered trademark), and the time until the rate of change of the base fluorescence intensity became stable at ± 2%. The odor detection kit according to the example to which no odor molecule was added was placed on a fluorometer, and the rate of change in the base fluorescence intensity was ± 2%, which was compared with the time until it became stable. As a result, as shown in FIG. 18, the time until stabilization was 300 ± 30 seconds for the suspension of the odor detection cells and 70 ± 6 seconds for the odor detection kit according to the example. Therefore, it was shown that the odor detection kit according to the example in which the odor detection cells are arranged in a single layer on the substrate has a shorter time to stabilize.

(Example 13: Comparison of responsiveness between cell suspension and kit)
In order to compare the response of the odor detection cell suspension to the odor molecule and the response of the odor detection kit according to the example to the odor molecule, each of the assay buffer (control) and geosmin 10 μmol / L was used. The rate of change in fluorescence intensity 120 seconds after the addition was measured with a fluorometer (Quantus, Fluorometer, registered trademark). As a result, as shown in FIG. 19, in the case of the suspension of odor-detecting cells, the rate of change in fluorescence intensity after the addition of the control was -3.9 ± 6.5%, and the rate of change in the fluorescence intensity after the addition of geosmin was It was 3.2 ± 3.6%. In the case of the odor detection kit according to the example, the rate of change in fluorescence intensity after the addition of the control was 2.4 ± 1.7%, and the rate of change in the fluorescence intensity after the addition of geosmin was 27.5 ± 4.3%. .. Therefore, it was shown that when the odor detection kit according to the example was used, the rate of change in fluorescence intensity when the control was added was small, and the rate of change in fluorescence intensity when the odor molecule was added was large.

(Example 14: Comparison of variation in measurement results between cell suspension and kit)
Based on the results shown in FIG. 19, the absolute value of the coefficient of variation of the rate of change of each fluorescence intensity was obtained. The absolute value of the coefficient of variation represents the variation. As a result, as shown in FIG. 20, in the case of the suspension of odor detection cells, the absolute value of the coefficient of variation of the change rate of the fluorescence intensity after the addition of the control was 165.9%, and the change rate of the fluorescence intensity after the addition of geosmin. The absolute value of the coefficient of variation of was 113.8%. In the case of the odor detection kit according to the example, the absolute value of the coefficient of variation of the rate of change in fluorescence intensity after the addition of the control is 71.2%, and the absolute value of the coefficient of variation of the coefficient of variation of the rate of change in fluorescence intensity after the addition of geosmin is 15.5. %Met. Therefore, it was shown that the variation in the measurement results was small when the odor detection kit according to the example was used.

(Example 15: Measurement of water obtained from a dam lake)
At the shore of the dam lake, the assay buffer alone (control) was added to the kit and geosmin 100 μmol / L was added using the odor detection kit and the fluorometer (registered trademark) according to the examples. The rate of change in fluorescence intensity when the assay buffer was added to the kit was measured. As a result, as shown in FIG. 21, when the assay buffer alone (control) was added to the kit, the rate of change in fluorescence intensity was 2.8%, and the assay buffer containing 100 μmol / L of geosmin was added to the kit. In the case, the rate of change in fluorescence intensity was 21.2%. Therefore, it was shown that the odor molecule can be detected even outdoors by using the odor detection kit according to the example.

Next, at the shore of the dam lake, when the surface water of the dam water was added to the kit using the odor detection kit and the fluorometer (registered trademark) according to the examples, and geosmin 100 μmol / L was added. The rate of change in fluorescence intensity when the surface water of the added dam water was added to the kit was measured. As a result, as shown in FIG. 21, when the surface water of the dam water alone was added to the kit, the rate of change in the fluorescence intensity was 6.0%, and the surface water of the dam water to which 100 μmol / L of geosmin was added was added to the kit. When added to, the rate of change in fluorescence intensity was 13.7%. When the surface water of the dam water was analyzed by GC / MS, the surface water of the dam water contained 300 pmol / L of geosmin. Therefore, it was shown that by using the odor detection kit according to the examples, it is possible to detect specific odor molecules even when dam lake water containing background odors and contaminants is inspected.

(Example 16: Preparation of DmOr56a receptor and DmOr82a receptor-expressing cells)
A homogeneous odor detection cell line was prepared from Sf21 cells expressing DmOr56a receptor and co-receptor DmOrco, and GCaMP6s by the method described in Example 5.

In addition, the start codon to the stop codon of the receptor DmOr82a gene derived from the tactile cDNA of Drosophila melanogaster was amplified with a primer containing the following gene-specific sequence, and the pIEx4 vector (Novagen) multicloning site (Novagen) It was inserted into BamHI, NotI) to construct a pIEx4-DmOr82a vector.
DmOr82a
Forward; 5'-ACGTCAAGAGCTCGCGGATCGCCGCCATGGGTAGGCTGTTTCAACTG-3'
Reverse; 5'-CTTTACCAGAAGATGCGGCCTTACTCAATAGATTTGATGAG-3'

The constructed pIEx4-DmOr82a vector is received by the lipofection method (TransIT-insect; Mirus, registered trademark) together with the pIE1-neo vector (Novagen; G418 resistance gene) according to the attached manual of TransIT-Insect. The gene was introduced into a homogeneous odor detection cell line expressing the body. As a result, cells expressing the DmOr82a receptor in addition to the DmOr56a receptor were prepared.

(Example 17: Preparation of DmOr56a receptor and DmOr49b receptor-expressing cells)
A homogeneous odor detection cell line was prepared from Sf21 cells expressing DmOr56a receptor and co-receptor DmOrco, and GCaMP6s by the method described in Example 5.

Further, the start codon to the stop codon of the G418 resistance gene derived from the pIE1-neo vector is amplified with a primer containing the following gene-specific sequence, and the pIZ vector (pIZ / V5-His (manufactured by Invitrogen)) is multicloned. It was inserted into the site (HindIII, SacII) to construct a pIZ-neo vector.
G418 (neo)
Forward; 5'-ATCTGTTCGAATTTAAAGCTGCCGCCATGATTGAACAAGATGGATTGC-3'
Reverse; 5'-GATAGGCTTACCTTCGAACCTCAGAAGAACTCGTCAAGAAG-3'

Next, the protein expression cassette of the pIZ-neo vector (the portion linked to the OpIE2 promoter (P (OpIE2)), neo gene, and OpIE2 polyA addition signal (OpIE2pA)) was amplified and amplified at the Pci1 site of the pIB vector. A protein expression cassette was inserted to construct a G418 resistance gene insertion vector, pIB-neo vector.

Next, the start codon to the stop codon of the receptor DmOr49b gene derived from the tactile cDNA of Drosophila melanogaster was amplified with a primer containing the following gene-specific sequence, and the pIB-neo vector (above) was mulched. It was inserted into a cloning site (EcoRI, XhoI) to construct a pIB-DmOr49b-neo vector.
DmOr49b
Forward; 5'-CAGTGTGGTGGAATTGCCGCCATGTTTGAAGACATTCAGCTAATC-3'
Reverse; 5'-GCCCTCTAGACTCGATCATCCGTAGACTCGCTTG-3'

The constructed pIB-DmOr49b-neo vector was applied to a homogeneous odor detection cell line expressing the DmOr56a receptor by the lipofection method (TransIT-insect; registered trademark of Mirus) according to the attached manual of TransIT-Insect. The gene was introduced. As a result, cells expressing the DmOr49b receptor in addition to the DmOr56a receptor were prepared.

(Example 18: Reactivity of DmOr56a receptor and DmOr82a receptor expressing cells)
Cells expressing the DmOr56a receptor and the DmOr82a receptor were prepared by the method described in Example 16.

In addition, a solution containing no odor (negative control), a solution containing geranyl acetate at a concentration of 300 μmol / L, and a solution containing geosmin at a concentration of 10 μmol / L were prepared. Geranyl acetate is the target odor of the DmOr82a receptor. Geosmin is the target odor of the DmOr56a receptor. As shown in FIG. 22, the negative control solution, the solution containing geranyl acetate, and the solution containing geosmin were contacted with the cells expressing the DmOr56a receptor and the DmOr82a receptor in this order. As a result, the fluorescence intensity of the cells expressing the DmOr56a receptor and the DmOr82a receptor changed by 23.2% with respect to geranyl acetate and 16.4% with respect to geosmin.

(Example 19: Reactivity of DmOr56a receptor and DmOr49b receptor expressing cells)
Cells expressing the DmOr56a receptor and the DmOr49b receptor were prepared by the method described in Example 17.

In addition, a solution containing no odor (negative control), a solution containing 2-methylphenol at a concentration of 300 μmol / L, and a solution containing geosmin at a concentration of 10 μmol / L were prepared. 2-Methylphenol is the target odor of the DmOr49b receptor. Geosmin is the target odor of the DmOr56a receptor. As shown in FIG. 23, the negative control solution, the solution containing 2-methylphenol, and the solution containing geosmin were contacted with the cells expressing the DmOr56a receptor and the DmOr49b receptor in this order. As a result, the fluorescence intensity of the cells expressing the DmOr56a receptor and the DmOr49b receptor changed by 37.6% with respect to 2-methylphenol and by 29.4% with respect to geosmin.

(Example 20: Concentration-dependent reactivity of DmOr56a receptor and DmOr82a receptor-expressing cells)
Cells expressing the DmOr56a receptor and the DmOr82a receptor were prepared by the method described in Example 16.

Further, the assay buffer (140 mmol / L) was prepared so that the final concentration of geranyl acetate was 0 mol / L (control), 1 μmol / L, 3 μmol / L, 10 μmol / L, 30 μmol / L, 100 μmol / L, and 300 μmol / L. NaCl, 5.6 mmol / L KCl, 4.5 mmol / L CaCl2, 11.26 mmol / L MgCl2, 10 mmol / L HEPES, 9.4 mmol / L D-glucose, pH 7.2). did. Also, as a positive control, geosmin was diluted in assay buffer to a final concentration of 10 μmol / L. All solutions were prepared to contain DMSO at a final concentration of 0.1%.

Next, as shown in FIG. 24, each of the solutions was contacted with cells expressing the DmOr56a receptor and the DmOr82a receptor at 6-minute intervals. As a result, as shown in FIGS. 24 and 25, the cells expressing the DmOr56a receptor and the DmOr82a receptor emitted intense fluorescence according to the concentration of geranyl acetate. It also fluoresces against the positive control geosmin.

(Example 21: Concentration-dependent reactivity of DmOr56a receptor and DmOr49b receptor-expressing cells)
Cells expressing the DmOr56a receptor and the DmOr49b receptor were prepared by the method described in Example 17.

In addition, 2-methylphenol is stepped in assay buffer so that the final concentration is 0 mol / L (control), 1 μmol / L, 3 μmol / L, 10 μmol / L, 30 μmol / L, 100 μmol / L, and 300 μmol / L. Diluted. Also, as a positive control, geosmin was diluted in assay buffer to a final concentration of 10 μmol / L. All solutions were prepared to contain DMSO at a final concentration of 0.1%.

Next, as shown in FIG. 26, each of the solutions was contacted with cells expressing the DmOr56a receptor and the DmOr49b receptor at 6-minute intervals. As a result, as shown in FIGS. 26 and 27, the cells expressing the DmOr56a receptor and the DmOr49b receptor emitted intense fluorescence according to the concentration of 2-methylphenol. It also fluoresces against the positive control geosmin.

(Example 22: Specific reactivity of olfactory receptor-expressing cells)
The cells expressing the DmOr56a receptor were prepared by the method described in Example 5, and the cells expressing the DmOr56a receptor and the DmOr82a receptor were prepared by the method described in Example 16. Cells expressing the DmOr56a receptor and the DmOr49b receptor were prepared by the method described in 17.

In addition, a solution containing no odor (negative control), a solution containing benzaldehyde at a concentration of 300 μmol / L, a solution containing methyl benzoate at a concentration of 300 μmol / L, and 1-octen-3-ol at a concentration of 300 μmol / L. A solution containing 2-methylphenol at a concentration of 300 μmol / L, a solution containing geranyl acetate at a concentration of 300 μmol / L, and a solution containing geosmine at a concentration of 10 μmol / L were prepared.

When each solution was brought into contact with cells expressing the DmOr56a receptor, as shown in FIG. 28, the fluorescence intensity of the cells expressing the DmOr56a receptor changed by 68.1% with respect to geosmin. The fluorescence intensity of cells expressing the DmOr56a receptor and the DmOr82a receptor changed 7.4% with respect to benzaldehyde, 34.5% with respect to geranyl acetate, and 4.3% with respect to geosmin. The fluorescence intensity of cells expressing the DmOr56a and DmOr49b receptors changed 19.4% with respect to benzaldehyde, 58.5% with respect to 2-methylphenol, and 29.6% with respect to geosmin. ..

10 ... substrate, 20 ... odor detection cells, 30 ... tubes, 40 ... fluid

Claims

With the board
An odor detection cell having an olfactory receptor and expressing a fluorescent protein whose fluorescence intensity changes according to the concentration of an odor molecule, which is arranged in a single layer on the substrate.
An odor detection kit for fluorometers.
The odor detection kit according to claim 1, wherein the odor detection cells are fixed to the substrate via a cell membrane modifier.
The odor detection kit according to claim 1 or 2, further comprising a tube capable of fixing the substrate inside.
The odor detection kit according to any one of claims 1 to 3, wherein the odor detection cell expresses an olfactory receptor by a transgene.
The odor detection kit according to any one of claims 1 to 4, wherein the odor detection cells express an insect olfactory receptor.
The odor detection kit according to any one of claims 1 to 5, wherein the olfactory receptor is an ion channel type receptor.
The odor detection kit according to claim 6, wherein the fluorescent protein changes the fluorescence intensity according to the ion concentration.
The odor detection kit according to any one of claims 1 to 7, wherein the olfactory receptor is selected from BmOR1, BmOR3, DmOr13a, DmOr56a, DmOr82a, DmOr49b, DmOr85b and PxOR1.
The odor detection kit according to any one of claims 1 to 8, wherein the odor detection cell is an insect cell.
The odor detection kit according to claim 9, wherein the insect cell is a cell derived from ga.
The odor detection kit according to claim 9 or 10, wherein the insect cells are selected from Sf21, Sf9, High Five, and Tni-derived cells.
The odor detection kit according to any one of claims 9 to 11, wherein the insect cell is a cell derived from Drosophila.
The odor detection kit according to any one of claims 9 to 12, wherein the insect cell is a Drosophila S2 cell.
The odor detection kit according to any one of claims 1 to 13, wherein the substrate is transparent.
To prepare odor detection cells that have olfactory receptors and express fluorescent proteins whose fluorescence intensity changes according to the concentration of odor molecules.
Preparing the board and
By arranging the odor detection cells in a single layer on the substrate,
A method of manufacturing an odor detection kit for a fluorometer, including.