Method for measuring response of olfactory receptor to substance

Abstract

Provided is a method for measuring the response of an olfactory receptor to a substance. The response of an olfactory receptor to a test substance is measured by bringing the test substance into contact with the olfactory receptor under conditions where the response of an adenosine receptor to the test substance is reduced.

Description

Method for measuring olfactory receptor responses to substances

[0001] The present invention relates to a method for measuring the response of olfactory receptors to a substance.

[0002] Aroma is an important factor that influences the palatability of foods, cosmetics, etc. Therefore, techniques for screening aroma components necessary for reproducing a specific aroma, techniques for reproducing a specific aroma by combining aroma components, and techniques for screening components that suppress (i.e., mask) a specific undesirable aroma are industrially important techniques for developing foods, cosmetics, etc.

[0003] Conventionally, screening for compounds that affect specific aromas has been carried out by humans evaluating the aroma of test substances through sensory testing, but sensory testing has problems such as the need to train experts who can evaluate aromas and low throughput.

[0004] In mammals such as humans, odors are perceived when molecules of odorant components bind to olfactory receptors on olfactory nerve cells present in the olfactory epithelium in the upper part of the nasal cavity, and the receptor's response to the molecules is transmitted to the central nervous system. Specifically, when activated by an odorant, olfactory receptors are known to couple with intracellular G proteins (specifically, Gs such as Golf) to activate adenylate cyclase, thereby increasing the amount of intracellular cAMP (Non-Patent Document 1). In recent years, methods have been reported for screening substances that exhibit or suppress specific odors using the response of olfactory receptors as an indicator (Patent Documents 1 to 4, etc.).

[0005] Adenosine receptors are G protein-coupled receptors (GPCRs) that respond to adenosine. Known adenosine receptors include adenosine receptors A1, A2A, A2B, and A3. Adenosine receptors A2A and A2B are known to be Gs protein-coupled adenosine receptors.

[0006] Patent Publication No. 2019-037197, WO2021 / 064201, Patent Publication No. 2019-129773, Patent Publication No. 2019-129772

[0007] Kajiya K. et al., Molecular bases of odor discrimination: Reconstitution of olfactory receptors that recognize overlapping sets of odorants. Journal of Neuroscience, 2001, 21:6018-6025

[0008] An objective of the present invention is to provide a method for measuring the response of olfactory receptors to a substance.

[0009] The present inventors discovered that when measuring the response of olfactory receptors to a test substance such as food, non-specific responses (i.e., responses other than the response of olfactory receptors to the test substance) can be observed, and that the use of an adenosine receptor antagonist can reduce such non-specific responses, thereby enabling the response of olfactory receptors to the test substance to be specifically measured, and thus completed the present invention.

[0010] That is, the present invention can be exemplified as follows. [1] A method for measuring the response of an olfactory receptor to a test substance, comprising the following steps (A) and (B): (A) contacting an olfactory receptor with a test substance; and (B) measuring the response of the olfactory receptor to the test substance, wherein the olfactory receptor is used in a form supported on a cell or in a form supported on a cell membrane prepared from the cell, and step (A) is performed under the following condition (X): (X) a condition in which the response of the adenosine receptor to the test substance is reduced. [2] The method wherein the adenosine receptor is adenosine receptor A2A and / or A2B. [3] The method wherein step (A) is performed in the presence of an adenosine receptor antagonist, thereby achieving condition (X). [4] The method, wherein the adenosine receptor antagonist is selected from the group consisting of caffeine, theophylline, istradefylline, CGS-15943, SCH-58261, SCH-442416, ZM-241385, CVT-6883, MRS-1706, MRS-1754, MRE-2029-F20, PSB-603, PSB-0788, and PSB-1115. [5] The method, wherein the adenosine receptor antagonist is used at a concentration of 0.05 to 1000 μM. [6] The method, wherein the condition (X) is achieved by the cells having the following characteristic (X1) or (X2): (X1) the cells have been modified to reduce the activity of the adenosine receptor; or (X2) the cells do not naturally have the adenosine receptor. [7] The method, wherein the cell has been modified so as not to have the adenosine receptor. [8] The method, wherein the response of the olfactory receptor is activation of the olfactory receptor. [9] The method, wherein the response of the olfactory receptor is inactivation of the olfactory receptor, and step (A) is carried out in the presence of a substance that activates the olfactory receptor.

[10] The method, wherein the olfactory receptor is used in a form supported by the cell.

[11] The method, wherein the cell is an animal cell.

[12] The method, wherein the response is measured using intracellular cAMP concentration or intracellular calcium concentration as an index.

[13] The intracellular cAMP concentration is

[14] The method as described above, wherein the test substance is selected from the group consisting of foods, their raw materials, and fractions thereof.

[15] The method as described above, wherein the olfactory receptor is selected from the group consisting of OR1A1, OR1A2, OR1B1, OR1C1, OR1D2, OR1D5, OR1E1, OR1F1, OR1F12, OR1G1, OR1I1, OR1J1, OR1J2, OR1J4, OR1K1, OR1L1, OR1L3, OR1L4, OR1L8, OR1M1, OR1N1, OR1N2, OR1Q1, OR1R1P, OR1S1, OR2A1, OR2A2, OR2A4, OR2A5, OR2A12, OR2A14, OR2A25, OR2AE1, OR2AG1, OR2AG2, OR2AE3, OR2AG4, OR2AE5, OR2AG5, OR2AE6, OR2AG6, OR2AG7, OR2AE8, OR2AG8, OR2AE9, OR2AG9, OR2AG10, OR2AG11, OR2AG12, OR2A13, OR2A14, OR2A15, OR2AE1, OR2AG13, OR2AG14, OR2A15, OR2AE2, OR2AG15, OR2AG16, OR2AG17, OR2AG18, OR2AG19, OR2AG20, OR2A21, OR2A22, OR2A23, OR2AE19, OR2AG19, OR2AG21, OR2A24, OR2A25, OR2AE10, OR2AG11, OR2AG22, OR2A25, OR2AE11, OR2AG12, OR2AG23, OR2A25, OR2AE11, OR2AG13, OR2AG24, OR2A25, R2AJ1P, OR2AK2, OR2AP1, OR2AT4, OR2B2, OR2B3, OR2B6, OR2B11, OR2C1, OR2C3, OR2D2, OR2D3, OR2F1, OR2G2, OR2G3, OR2G6, OR2H1, OR2H2, O R2J2, OR2J3, OR2K2, OR2L2, OR2L8, OR2L13, OR2M2, OR2M4, OR2M7, OR2S2, OR2T1, OR2T2, OR2T5, OR2T6, OR2T8, OR2T10, OR2T11, OR2T27, OR2 T34, OR2V2, OR2W1, OR2W3, OR2Y1, OR2Z1, OR3A1, OR3A2, OR3A3, OR3A4, OR4A5, OR4A15, OR4A16, OR4A47, OR4B1, OR4C3, OR4C5, OR4C6, OR4C1 1, OR4C12, OR4C13, OR4C15, OR4C16, OR4C46, OR4D1, OR4D2, OR4D5, OR4D6, OR4D9, OR4D10, OR4D11, OR4E2, OR4F3, OR4F5, OR4F6, OR4F14P, O R4F15, OR4G11P, OR4H12P, OR4K1, OR4K2, OR4K5, OR4K13, OR4K14, OR4K15, OR4K17, OR4L1, OR4M1, OR4N2, OR4N4, OR4N5, OR4P4, OR4Q3, OR4S 1, OR4S2, OR4X1, OR4X2, OR5A1, OR5A2, OR5AC2, OR5AK2, OR5AK3P, OR5AN1, OR5AP2, OR5AR1, OR5AS1, OR5AU1, OR5B2, OR5B3, OR5B12, OR5B17,OR5B21、OR5C1、OR5D13、OR5D14、OR5D16、OR5D18、OR5F1、OR5H1、OR5H2、OR5H6、OR5H14、OR5I1、OR5J2、OR5K1、OR5K3、OR5K4、OR5L2、OR5M3、OR5M8、OR5M9、OR5M10、OR5M11、OR5P3、OR5R1、OR5T1、OR5T2、OR5T3、OR5V1、OR5W2、OR6A2、OR6B1、OR6B2、OR6C1、OR6C2、OR6C3、OR6C4、OR6C6、OR6C65、OR6C66P、OR6C68、OR6C70、OR6C74、OR6C75、OR6C76、OR6F1、OR6J1、OR6K2、OR6K3、OR6K6、OR6M1、OR6N1、OR6N2、OR6P1、OR6Q1、OR6S1、OR6T1、OR6V1、OR6X1、OR6Y1、OR7A3P、OR7A5、OR7A10、OR7A17、OR7C1、OR7C2、OR7D2、OR7D4、OR7E24、OR7G1、OR7G2、OR7G3、OR8A1、OR8B3、OR8B4、OR8B8、OR8B12、OR8D1、OR8D2、OR8D4、OR8G2、OR8G5、OR8H3、OR8I2、OR8J1、OR8J3、OR8K1、OR8K3、OR8K5、OR8S1、OR8U1、OR9A4、OR9G1、OR9G4、OR9I1、OR9K2、OR9Q1、OR9Q2、OR10A3、OR10A4、OR10A5、OR10A6、OR10A7、OR10AD1、OR10AG1、OR10C1、OR10D3、OR10D4P、OR10G2、OR10G3、OR10G4、OR10G6、OR10G7、OR10G9、OR10H2、OR10H4、OR10J1、OR10J3、OR10J5、OR10K1、OR10K2、OR10P1、OR10Q1、OR10R2、OR10S1、OR10T2、OR10V1、OR10W1、OR10X1、OR10Z1、OR11A1、OR11G2、OR11H4、OR11H6、OR11H12、OR11L1、OR12D2、OR12D3、OR13A1、OR13C2、OR13C3、OR13C4、OR13C8、OR13D1、OR13F1、OR13G1、OR13H1、OR13J1、OR14A2、OR14A16、OR14C36、OR14I1、OR14J1、OR14K1、OR14L1P, OR51A1P, OR51A4, OR51A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51D1, OR51E1, OR51E2, OR51F1, OR51F2, OR51F5P, OR51G 1, OR51G2, OR51H1, OR51I1, OR51I2, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4,

[16] The method, wherein the olfactory receptor is selected from the group consisting of OR52B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E8, OR52H1, OR52I2, OR52J3, OR52K2, OR52L2P, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52P2P, OR52R1, OR52W1, OR52Z1P, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B2P, and OR56B4.

[0011] Figure showing the response of control cells (cells transfected with the empty vector Rho-pME18S) to adenosine. Figure showing the suppression of the response of control cells (cells transfected with the empty vector Rho-pME18S) to adenosine by the adenosine receptor antagonist CGS-15943. Figure showing the response of control cells (cells transfected with the empty vector Rho-pME18S) to a test substance. Figure showing the response of cells expressing the olfactory receptor OR10G7 to a test substance. Figure showing the correlation between the adenosine concentration in the test substance and the non-specific response of cells to the test substance. Figure showing olfactory receptor activity (common logarithm of the normalized response) measured in the presence or absence of the adenosine receptor antagonist CGS-15943 using Hon-Dashi® dried sardine stock as the test substance. Panel (A): Measurement results in the absence of CGS-15943. Panel (B): Measurement results in the presence of CGS-15943. This figure shows olfactory receptor activity (common logarithm of normalized response) measured in the presence or absence of the adenosine receptor antagonist CGS-15943 using Knorr® Creamed Corn with Lots of Corn as the test substance. Panel (A): Measurement results in the absence of CGS-15943. Panel (B): Measurement results in the presence of CGS-15943.

[0012] The present invention will be described in detail below.

[0013] The method of the present invention is a method for measuring the response of an olfactory receptor to a test substance. The method of the present invention can be carried out under conditions in which the response of an adenosine receptor to a test substance is reduced. By carrying out the method of the present invention under conditions in which the response of an adenosine receptor to a test substance is reduced, the response of the olfactory receptor to the test substance can be specifically measured. Specifically, the method of the present invention can be carried out by contacting the test substance with an olfactory receptor under conditions in which the response of the adenosine receptor to the test substance is reduced, and measuring the response of the olfactory receptor to the test substance. The adenosine receptor may be present in a cell or cell membrane that carries the olfactory receptor. That is, the method of the present invention may specifically be a method for measuring the response of an olfactory receptor to a test substance, comprising: (A) a step of contacting the olfactory receptor with the test substance; and (B) a step of measuring the response of the olfactory receptor to the test substance, wherein the olfactory receptor is used in a form supported by a cell or a form supported by a cell membrane prepared from the cell, and step (A) is carried out under conditions in which the response of the adenosine receptor to the test substance is reduced.

[0014] <1> Test Substance "Test substance" refers to a substance for measuring the response of an olfactory receptor in the method of the present invention. The test substance is not particularly limited as long as it can induce an adenosine receptor response. Examples of adenosine receptor responses include adenosine receptor activation. The test substance may consist of a single component or a combination of two or more components. When the test substance consists of a combination of two or more components, the number of components constituting the combination and their composition ratios are not particularly limited. The test substance may be a known substance or a novel substance. The test substance may be a natural product or an artificial product. The test substance may be, for example, a compound library created using combinatorial chemistry technology. Examples of test substances include substances that may contain an adenosine receptor agonist such as adenosine, and substances that may be adenosine receptor agonists. Examples of test substances include, in particular, substances that may contain adenosine. That is, the test substance may be, for example, a substance that can contain adenosine, as described below. Specific examples of the test substance include foods, cosmetics, their raw materials, and fractions thereof (e.g., distillates). Foods also include beverages and seasonings. Examples of foods include beverages such as coffee drinks, black tea drinks, green tea drinks, dairy drinks, lactic acid bacteria drinks, soft drinks, carbonated drinks, fruit juice drinks, vegetable drinks, sports drinks, jelly drinks, powdered drinks, and alcoholic beverages; noodle soups such as udon soup, soba soup, somen soup, ramen soup, champon soup, and pasta sauce; dashi stocks such as Japanese dashi and chicken broth; condiments such as soy sauce, fish sauce, Chinese sauce, oyster sauce, dressing, miso, mayonnaise, tomato ketchup, and creaming powder; soups such as egg soup, wakame seaweed soup, shark fin soup, Chinese soup, consommé soup, curry soup, potage soup, corn soup, clear soup, and miso soup; and extracts such as beef extract, pork extract, and chicken extract. Note that a certain food may also be an ingredient of another food.Cosmetics, perfumes, and toiletries are examples of cosmetic products. Specific examples of test substances include alcohols, ketones, aldehydes, ethers, esters, hydrocarbons, sugars, organic acids, nucleic acids, amino acids, peptides, and various other organic or inorganic components. Specific examples of test substances include existing food additives. "Existing food additives" refer to substances that have already been approved for use as food additives. A single test substance may be used, or two or more test substances may be used in combination.

[0015] <2> Olfactory receptor The olfactory receptor is not particularly limited. As the olfactory receptor, one type of olfactory receptor may be used, or two or more types of olfactory receptors may be used in combination. When two or more types of olfactory receptors are used in combination, the response of the olfactory receptor to the test substance may be measured, for example, for each of the olfactory receptors individually. For example, the response of each olfactory receptor to the test substance can be measured individually using cells expressing each olfactory receptor.

[0016] The olfactory receptors are OR1A1, OR1A2, OR1B1, OR1C1, OR1D2, OR1D4, OR1D5, OR1E1, OR1E2, OR1F1, OR1F12, OR1G1, OR1I1, OR1J1, OR1J2, OR1J4, OR1K1, OR1L1, OR1L3, OR1L4, OR1L6, OR1L8, OR1M1, OR1N1, OR1N2, OR1Q1, OR1R1P, OR1S1, OR1S2, OR2A1, OR2A2, OR2A4, OR2A5, OR2A7, OR2A12, OR2A14, OR2A25, OR2AE1, and OR2AG. 1, OR2AG2, OR2AJ1P, OR2AK2, OR2AP1, OR2AT4, OR2B2, OR2B3, OR2B6, OR2B11, OR2C1, OR2C3, OR2D2, OR2D3, OR2F1, OR2F2, OR2G2, OR2G3, OR2G6, OR2H1, O R2H2, OR2J1P, OR2J2, OR2J3, OR2K2, OR2L2, OR2L3, OR2L5, OR2L8, OR2L13, OR2M2, OR2M3, OR2M4, OR2M5, OR2M7, OR2S2, OR2T1, OR2T2, OR2T3, OR2T4, OR2T 5, OR2T6, OR2T7, OR2T8, OR2T10, OR2T11, OR2T12, OR2T27, OR2T29, OR2T33, OR2T34, OR2T35, OR2V1, OR2V2, OR2W1, OR2W3, OR2Y1, OR2Z1, OR3A1, OR3A2, OR3A3, OR3A4, OR4A4P, OR4A5, OR4A15, OR4A16, OR4A47, OR4B1, OR4C3, OR4C5, OR4C6, OR4C11, OR4C12, OR4C13, OR4C15, OR4C16, OR4C45, OR4C46, OR4D1, OR4D2, OR4D5, OR4D6, OR4D9, OR4D10, OR4D11, OR4E2, OR4F3, OR4F4, OR4F5, OR4F6, OR4F14P, OR4F15, OR4F17, OR4F21, OR4G11P, OR4H12P, OR4K1, OR4K2, OR4K5, OR4K13, OR4K14, OR4K15, OR4K17, OR4L1, OR4M1, OR4M2, OR4N2, OR4N4, OR4N5, OR4P4, OR4Q3, OR4S1, OR4S2, OR4X1, OR4X2, OR5A1, OR5A2, OR5AC2,OR5AK2、OR5AK3P、OR5AN1、OR5AP2、OR5AR1、OR5AS1、OR5AU1、OR5B2、OR5B3、OR5B12、OR5B17、OR5B21、OR5C1、OR5D13、OR5D14、OR5D16、OR5D18、OR5F1、OR5H1、OR5H2、OR5H6、OR5H14、OR5H15、OR5I1、OR5J2、OR5K1、OR5K2、OR5K3、OR5K4、OR5L1、OR5L2、OR5M1、OR5M3、OR5M8、OR5M9、OR5M10、OR5M11、OR5P2、OR5P3、OR5R1、OR5T1、OR5T2、OR5T3、OR5V1、OR5W2、OR6A2、OR6B1、OR6B2、OR6B3、OR6C1、OR6C2、OR6C3、OR6C4、OR6C6、OR6C65、OR6C66P、OR6C68、OR6C70、OR6C74、OR6C75、OR6C76、OR6F1、OR6J1、OR6K2、OR6K3、OR6K6、OR6M1、OR6N1、OR6N2、OR6P1、OR6Q1、OR6S1、OR6T1、OR6V1、OR6X1、OR6Y1、OR7A3P、OR7A5、OR7A10、OR7A17、OR7C1、OR7C2、OR7D2、OR7D4、OR7E24、OR7G1、OR7G2、OR7G3、OR8A1、OR8B2、OR8B3、OR8B4、OR8B8、OR8B12、OR8D1、OR8D2、OR8D4、OR8G1、OR8G2、OR8G5、OR8H1、OR8H2、OR8H3、OR8I2、OR8J1、OR8J3、OR8K1、OR8K3、OR8K5、OR8S1、OR8U1、OR8U8、OR9A2、OR9A4、OR9G1、OR9G4、OR9I1、OR9K2、OR9Q1、OR9Q2、OR10A2、OR10A3、OR10A4、OR10A5、OR10A6、OR10A7、OR10AD1、OR10AG1、OR10C1、OR10D3、OR10D4P、OR10G2、OR10G3、OR10G4、OR10G6、OR10G7、OR10G8、OR10G9、OR10H1、OR10H2、OR10H3、OR10H4、OR10H5、OR10J1、OR10J3、OR10J5、OR10K1、OR10K2、OR10P1、OR10Q1、OR10R2、OR10S1、OR10T2、OR10V1、OR10W1、OR10X1, OR10Z1, OR11A1, OR11G2, OR11H1, OR11H2, OR11H4, OR11H6, OR11H12, OR11L1, OR12D2, OR12 D3, OR13A1, OR13C2, OR13C3, OR13C4, OR13C5, OR13C8, OR13C9, OR13D1, OR13F1, OR13G1, OR13H1, OR1 3J1, OR14A2, OR14A16, OR14C36, OR14I1, OR14J1, OR14K1, OR14L1P, OR51A1P, OR51A2, OR51A4, OR51 A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51D1, OR51E1, OR51E2, OR51F1, OR51F2, OR51F5P, OR51G1, OR 51G2, OR51H1, OR51I1, OR51I2, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8, OR52H1, OR52I1 , OR52I2, OR52J3, OR52K1, OR52K2, OR52L1, OR52L2P, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52P2P, OR52R1, OR52W1, OR52Z1P, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B2P, and OR56B4.

[0017] Olfactory receptors include OR1A1, OR1A2, OR1B1, OR1C1, OR1D2, OR1D5, OR1E1, OR1F1, OR1F12, OR1G1, OR1I1, OR1J1, OR1J2, OR1J4, OR1K1, OR1L1, OR1L3, OR1L4, OR1L8, OR1M1, OR1N1, OR1N2, OR1Q1, OR1R1P, OR1S1, OR2A1, OR2A2, OR2A4, OR2A5, OR2A12, OR2A14, OR2A25, OR2AE1, OR2AG1, OR2AG2, OR2AJ1P, OR2AK2, OR 2AP1, OR2AT4, OR2B2, OR2B3, OR2B6, OR2B11, OR2C1, OR2C3, OR2D2, OR2D3, OR2F1, OR2G2, OR2G3, OR2G6, OR2H1, OR2H2, OR2J2, OR2J3, OR2K2, OR2L2, OR2 L8, OR2L13, OR2M2, OR2M4, OR2M7, OR2S2, OR2T1, OR2T2, OR2T5, OR2T6, OR2T8, OR2T10, OR2T11, OR2T27, OR2T34, OR2V2, OR2W1, OR2W3, OR2Y1, OR2Z1, OR3 A1, OR3A2, OR3A3, OR3A4, OR4A5, OR4A15, OR4A16, OR4A47, OR4B1, OR4C3, OR4C5, OR4C6, OR4C11, OR4C12, OR4C13, OR4C15, OR4C16, OR4C46, OR4D1, OR4D 2, OR4D5, OR4D6, OR4D9, OR4D10, OR4D11, OR4E2, OR4F3, OR4F5, OR4F6, OR4F14P, OR4F15, OR4G11P, OR4H12P, OR4K1, OR4K2, OR4K5, OR4K13, OR4K14, OR4K 15, OR4K17, OR4L1, OR4M1, OR4N2, OR4N4, OR4N5, OR4P4, OR4Q3, OR4S1, OR4S2, OR4X1, OR4X2, OR5A1, OR5A2, OR5AC2, OR5AK2, OR5AK3P, OR5AN1, OR5AP2, OR5AR1, OR5AS1, OR5AU1, OR5B2, OR5B3, OR5B12, OR5B17, OR5B21, OR5C1, OR5D13, OR5D14, OR5D16, OR5D18, OR5F1, OR5H1, OR5H2, OR5H6, OR5H14, OR5I1,OR5J2、OR5K1、OR5K3、OR5K4、OR5L2、OR5M3、OR5M8、OR5M9、OR5M10、OR5M11、OR5P3、OR5R1、OR5T1、OR5T2、OR5T3、OR5V1、OR5W2、OR6A2、OR6B1、OR6B2、OR6C1、OR6C2、OR6C3、OR6C4、OR6C6、OR6C65、OR6C66P、OR6C68、OR6C70、OR6C74、OR6C75、OR6C76、OR6F1、OR6J1、OR6K2、OR6K3、OR6K6、OR6M1、OR6N1、OR6N2、OR6P1、OR6Q1、OR6S1、OR6T1、OR6V1、OR6X1、OR6Y1、OR7A3P、OR7A5、OR7A10、OR7A17、OR7C1、OR7C2、OR7D2、OR7D4、OR7E24、OR7G1、OR7G2、OR7G3、OR8A1、OR8B3、OR8B4、OR8B8、OR8B12、OR8D1、OR8D2、OR8D4、OR8G2、OR8G5、OR8H3、OR8I2、OR8J1、OR8J3、OR8K1、OR8K3、OR8K5、OR8S1、OR8U1、OR9A4、OR9G1、OR9G4、OR9I1、OR9K2、OR9Q1、OR9Q2、OR10A3、OR10A4、OR10A5、OR10A6、OR10A7、OR10AD1、OR10AG1、OR10C1、OR10D3、OR10D4P、OR10G2、OR10G3、OR10G4、OR10G6、OR10G7、OR10G9、OR10H2、OR10H4、OR10J1、OR10J3、OR10J5、OR10K1、OR10K2、OR10P1、OR10Q1、OR10R2、OR10S1、OR10T2、OR10V1、OR10W1、OR10X1、OR10Z1、OR11A1、OR11G2、OR11H4、OR11H6、OR11H12、OR11L1、OR12D2、OR12D3、OR13A1、OR13C2、OR13C3、OR13C4、OR13C8、OR13D1、OR13F1、OR13G1、OR13H1、OR13J1、OR14A2、OR14A16、OR14C36、OR14I1、OR14J1、OR14K1、OR14L1P、OR51A1P、OR51A4、OR51A7、OR51B2、OR51B4、OR51B5、OR51B6、OR51D1、OR51E1、OR51E2, OR51F1, OR51F2, OR51F5P, OR51G1, OR51G2, OR51H1, OR51I1, OR51I2, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D1, OR52E2, OR52E4, O R52E5, OR52E8, OR52H1, OR52I2, OR52J3, OR52K2, OR52L2P, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52P2P, OR52R1, OR52W1, OR52Z1P, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B2P, and OR56B4.

[0018] Genes that encode olfactory receptors are also called "olfactory receptor genes."

[0019] Olfactory receptor genes and olfactory receptors include those of various organisms. Examples of organisms include animals such as mammals. Specific examples of mammals include Homo sapiens (humans), Mus musculus (mice), Rattus norvegicus (rat), Canis lupus familiaris (dogs), Felis catus (cats), Bos taurus (cattle), Sus scrofa (pigs), Pan troglodytes (chimpanzees), Macaca fascicularis (cyn-eating monkeys), and Equus caballus (horses). Mammals, in particular, include humans. The nucleotide sequences of olfactory receptor genes and amino acid sequences of olfactory receptors of various organisms can be obtained from public databases such as NCBI and Ensembl.

[0020] The olfactory receptor may be, for example, a protein having the known or naturally occurring amino acid sequence of the olfactory receptor described above. The olfactory receptor may also be, for example, a conservative variant of a protein having the known or naturally occurring amino acid sequence of the olfactory receptor described above. That is, the olfactory receptors identified by the above names encompass, for example, proteins having the known or naturally occurring amino acid sequence of the olfactory receptor identified by that name, and conservative variants thereof. A "conservative variant" refers to a variant that maintains the original function. With regard to an olfactory receptor, "maintaining the original function" may mean that the response of the olfactory receptor variant is elicited by a substance that elicits the response of the original olfactory receptor (e.g., a substance that activates the original olfactory receptor). Unless otherwise specified, the expression "a protein has an amino acid sequence" means that the protein contains the amino acid sequence, and also encompasses cases where the protein consists of the amino acid sequence. Variants include proteins having an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and / or added at one or several positions in a known or naturally occurring amino acid sequence. Specifically, "one or several" may mean, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3. Variants also include proteins having an amino acid sequence that is, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire known or naturally occurring amino acid sequence. Note that olfactory receptors identified by their originating biological species are not limited to the olfactory receptors themselves found in the biological species, but also include proteins having the amino acid sequence of the olfactory receptors found in the biological species, as well as variants thereof. Variants may or may not be found in the biological species. That is, for example, "human olfactory receptor" is not limited to the olfactory receptor itself found in humans, but also includes proteins having the amino acid sequence of the olfactory receptor found in humans and variants thereof.The olfactory receptor may be, for example, a chimeric protein of two or more olfactory receptors of different origins. That is, the olfactory receptors specified by the above names also encompass, for example, chimeric proteins of two or more olfactory receptors of different origins specified by the same names.

[0021] The "identity" between amino acid sequences refers to the identity between amino acid sequences calculated using blastp with default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment).

[0022] The olfactory receptor is used in a form supported on a cell or in a form supported on a cell membrane prepared from the cell.

[0023] A cell having an olfactory receptor is also referred to as a "cell of the present invention." An olfactory receptor can function by being localized, for example, in the cell membrane. Thus, a cell of the present invention may have an olfactory receptor, for example, in the cell membrane.

[0024] Olfactory receptors are expressed from olfactory receptor genes. Thus, the cells of the present invention have an olfactory receptor gene. Specifically, the cells of the present invention have an olfactory receptor gene in an expressible manner. It is sufficient for the cells of the present invention to have the olfactory receptor gene until the olfactory receptor is expressed. In other words, the cells of the present invention may or may not have the olfactory receptor gene after the olfactory receptor is expressed. In other words, the cells of the present invention are cells that have expressed an olfactory receptor gene, and are also cells that have expressed an olfactory receptor. It is to be noted that "expression of an olfactory receptor gene" and "expression of an olfactory receptor" can be used synonymously.

[0025] The cells of the present invention may have one copy of the olfactory receptor gene, or may have two or more copies of the olfactory receptor gene.

[0026] The cells of the present invention may inherently have an olfactory receptor gene, or may be modified to have an olfactory receptor gene.

[0027] Cells that inherently have an olfactory receptor gene include cells of organisms from which the above-mentioned olfactory receptor genes are derived, such as taste cells of mammals such as humans. Cells that inherently have an olfactory receptor gene can be obtained, for example, from organisms or tissues that contain the cells.

[0028] Cells modified to have an olfactory receptor gene include cells into which an olfactory receptor gene has been introduced.

[0029] The cells of the present invention and cells used to obtain them (for example, cells into which or into which an olfactory receptor gene is introduced) are also collectively referred to as "host cells."

[0030] The host cell is not particularly limited as long as it can express a functional olfactory receptor and can be used to measure the response of the olfactory receptor to a test substance. Examples of host cells include bacterial cells, fungal cells, plant cells, insect cells, and animal cells. Preferred host cells include eukaryotic cells such as fungal cells, plant cells, insect cells, and animal cells. More preferred host cells include animal cells. Examples of animals include mammals, birds, and amphibians. Examples of mammals include rodents and primates. Examples of rodents include Chinese hamsters, hamsters, mice, rats, and guinea pigs. Examples of primates include humans, monkeys, and chimpanzees. Examples of birds include chickens. Examples of amphibians include Xenopus laevis. Furthermore, the tissue or cell from which the host cell is derived is not particularly limited. Examples of tissues or cells from which host cells are derived include ovaries, kidneys, adrenal glands, tongue epithelium, olfactory epithelium, pineal gland, thyroid gland, and melanocytes. Examples of Chinese hamster cells include Chinese hamster ovary-derived cell lines (CHO). Specific examples of CHO include CHO-DG44 and CHO-K1. Examples of human cells include human embryonic kidney (HEK) cell lines. Specific examples of HEK include HEK293 and HEK293T. Examples of monkey cells include African green monkey kidney cell-derived cell lines (COS). Specific examples of COS include COS-1. Examples of Xenopus cells include Xenopus oocytes. Examples of insect cells include Spodoptera frugiperda-derived cells such as Sf9, Sf21, and SF+, and Trichoplusia ni-derived cells such as High-Five. The host cells may be individual independent cells (for example, free cells) or may form aggregates such as tissues.

[0031] Olfactory receptor genes can be obtained by cloning from organisms that have the olfactory receptor genes. Nucleic acids such as genomic DNA and cDNA containing the genes can be used for cloning. Olfactory receptor genes can also be obtained by chemical synthesis (Gene, 60(1), 115-127 (1987)).

[0032] The obtained olfactory receptor gene can be used as is or after appropriate modification. That is, by modifying the olfactory receptor gene, its variant can be obtained. Gene modification can be performed by known techniques. For example, a desired mutation can be introduced into a target site in DNA by site-directed mutagenesis. That is, for example, site-directed mutagenesis can be used to modify the coding region of a gene so that the encoded protein contains substitutions, deletions, insertions, and / or additions of amino acid residues at specific sites. Site-directed mutagenesis methods include PCR-based methods (Higuchi, R., 61, in PCR technology, Erlich, H.A. Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, H.J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T.A. et al., Meth. in Enzymol., 154, 367 (1987)). Variants of olfactory receptor genes can also be obtained directly by chemical synthesis.

[0033] The manner in which an olfactory receptor gene is introduced into a host cell is not particularly limited. The olfactory receptor gene may be expressibly maintained in the host cell. Specifically, for example, when an olfactory receptor gene is introduced in a form requiring transcription of DNA or the like, the olfactory receptor gene may be expressibly maintained in the host cell under the control of a promoter that functions in the host cell. In the host cell, the olfactory receptor gene may be present extrachromosomally or may be introduced onto the chromosome. When two or more genes are introduced, each gene may be expressibly maintained in the host cell.

[0034] The promoter for expressing the olfactory receptor gene is not particularly limited as long as it functions in the host cell. A "promoter functional in the host cell" refers to a promoter that has promoter activity in the host cell. The promoter may be a promoter native to the host cell or a heterologous promoter. The promoter may be the native promoter of the olfactory receptor gene or a promoter of another gene. The promoter may be stronger than the native promoter of the olfactory receptor gene. For example, promoters that function in animal cells include the SV40 promoter, EF1a promoter, RSV promoter, CMV promoter, and SRalpha promoter. Furthermore, highly active versions of native promoters may be obtained and used by using various reporter genes. Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

[0035] An olfactory receptor gene can be introduced into a host cell using, for example, a vector containing the gene. A vector containing an olfactory receptor gene is also referred to as an "olfactory receptor gene expression vector." An olfactory receptor gene expression vector can be constructed, for example, by ligating a DNA fragment containing the olfactory receptor gene to a vector. The olfactory receptor gene expression vector can be introduced into a host cell by introducing the olfactory receptor gene into the host cell. The vector may comprise a marker such as a drug resistance gene. The vector may also comprise an expression regulatory sequence such as a promoter for expressing the inserted gene. The vector can be selected appropriately depending on various conditions, such as the type of host cell and the mode of introduction of the olfactory receptor gene. For example, vectors that can be used to introduce genes into animal cells include plasmid vectors and viral vectors. Examples of viral vectors include retroviral vectors and adenoviral vectors. Examples of plasmid vectors include pcDNA series vectors (pcDNA3.1, etc.; Thermo Fisher Scientific), pBApo-CMV series vectors (Takara Bio), and pCI-neo (Promega). Depending on the type and configuration of the vector, the vector can be integrated into the chromosome of the host cell, can autonomously replicate extrachromosomally, or can be temporarily maintained extrachromosomally in the host cell. For example, vectors having a viral replication origin such as the SV40 replication origin can autonomously replicate extrachromosomally in animal cells. Specifically, for example, the pcDNA series vectors have the SV40 replication origin and can autonomously replicate extrachromosomally in host cells (e.g., COS-1 and HEK293T) that express the SV40 large T antigen.

[0036] Alternatively, the olfactory receptor gene can be introduced into a host cell by, for example, introducing a nucleic acid fragment containing the gene into the host cell. A nucleic acid fragment containing the olfactory receptor gene is also referred to as an "olfactory receptor gene fragment." Such fragments include linear DNA and linear RNA. Examples of linear RNA include mRNA and cRNA.

[0037] The method for introducing nucleic acids such as vectors and nucleic acid fragments into host cells can be selected appropriately depending on various conditions such as the type of host cells. For example, methods for introducing nucleic acids such as vectors and nucleic acid fragments into host cells such as animal cells include the DEAE-dextran method, calcium phosphate method, lipofection, electroporation, and microinjection. Furthermore, when the vector is a viral vector, the vector can be introduced into the host cells by infecting the host cells with the vector (virus).

[0038] Alternatively, cells inherently containing an olfactory receptor gene may be modified to increase expression of the olfactory receptor gene. "Increased gene expression" means that the expression level of the gene per cell is increased compared to unmodified cells. "Unmodified cells" herein refer to control cells that have not been modified to increase expression of the target gene. Examples of unmodified cells include wild-type cells and the original cells. Techniques for increasing expression of an olfactory receptor gene include increasing the copy number of the olfactory receptor gene and improving the transcription efficiency or translation efficiency of the olfactory receptor gene. The copy number of the olfactory receptor gene can be increased by introducing the olfactory receptor gene into host cells. Introduction of the olfactory receptor gene can be carried out as described above. The introduced olfactory receptor gene may be derived from the host cell or from a heterologous source. The transcription efficiency or translation efficiency of the olfactory receptor gene can be improved by modifying the gene expression regulatory sequence, such as a promoter. For example, the transcription efficiency of the olfactory receptor gene can be improved by replacing the promoter of the olfactory receptor gene with a stronger promoter.

[0039] The cells of the present invention may have any other properties as long as they can be used to measure the response of olfactory receptors to a test substance. Such properties include, for example, properties that are useful for measuring the response of olfactory receptors to a test substance.

[0040] The cells of the present invention may or may not have, for example, olfactory receptors other than the selected olfactory receptor (also referred to as "other olfactory receptors"). It may be preferable that the cells of the present invention do not have other olfactory receptors. Examples of cells that do not have other olfactory receptors include cells that do not have genes encoding other olfactory receptors, and cells that have genes encoding other olfactory receptors but do not express said genes. The cells of the present invention may, for example, not inherently have other olfactory receptors, or may be modified so that they do not have other olfactory receptors. Modifying cells so that they do not have other olfactory receptors can be achieved, for example, by knocking out genes encoding other olfactory receptors.

[0041] Furthermore, the cells of the present invention may contain, for example, a protein involved in signal transduction. In other words, the cells of the present invention may contain a gene encoding a protein involved in signal transduction. Examples of proteins involved in signal transduction include G proteins (e.g., Golf), G protein activators (e.g., Ric8B), adenylate cyclase, and calcium channels. Examples of Golf include animal Golf such as human Golf (GenBank accession No. NP_892023). Examples of Ric8B include animal Ric8B such as rat Ric8B (GenBank accession No. NP_783188). The cells of the present invention may contain, for example, components corresponding to the parameter to be measured. Examples of such components include a probe such as a calcium indicator and a reporter gene such as a luciferase gene. When a probe such as a calcium indicator is expressed from a gene, the cells of the present invention may contain a gene encoding the probe.

[0042] Furthermore, the cells of the present invention may contain, for example, a protein that promotes membrane expression of an olfactory receptor. In other words, the cells of the present invention may contain a gene encoding such a protein. Examples of such proteins include RTP1s (Zhuang H and Matsunami H, J Biol Chem 282, 15284-15293 (2007)). Examples of RTP1s include animal RTP1s such as human RTP1s (GenBank accession No. AAT70680), mouse RTP1s (GenBank accession No. ABU23737), and bat RTP1s (the amino acid sequence from the methionine residue at position 37 to the C-terminus of GenBank accession No. XP_006765914). Mouse RTP1s shares 93.3% amino acid identity with the human RTP1s. Bat RTP1s (the partial sequence described above) shares 90.7% amino acid identity with the human RTP1s.

[0043] The cells of the present invention may inherently possess the properties exemplified above, or may be modified to have the properties exemplified above. Regarding cell modification, the description of cell modification related to olfactory receptor genes, such as the introduction of olfactory receptor genes, can be applied mutatis mutandis. The genes exemplified above may be genes derived from the host cell or genes derived from a different species. Furthermore, the genes exemplified above may or may not be derived from the same source as the olfactory receptor gene. When two or more genes are introduced, it is sufficient that each gene is retained in the host cell in an expressible manner. For example, all of the genes may be retained on a single expression vector, or all of the genes may be retained on a chromosome. Furthermore, the genes may be retained separately on multiple expression vectors, or may be retained separately on a single or multiple expression vectors and on a chromosome. The genes exemplified above and the proteins encoded thereby may have, for example, the nucleotide sequences and amino acid sequences of known genes and proteins, respectively. Furthermore, the genes exemplified above and the proteins encoded thereby may be, for example, conservative variants of known genes and proteins, respectively. For conservative variants of genes and proteins, the descriptions regarding conservative variants of olfactory receptor genes and olfactory receptors can be applied mutatis mutandis.

[0044] The cells of the present invention may have the following characteristics (X1) or (X2): (X1) the cells have been modified to reduce the activity of adenosine receptors; (X2) the cells do not naturally have adenosine receptors.

[0045] Examples of cells having the characteristic (X1) include cells modified so as not to have adenosine receptors. That is, the characteristic (X1) includes cells modified so as not to have adenosine receptors. In other words, the cells of the present invention may be cells that do not have adenosine receptors, such as cells modified so as not to have adenosine receptors or cells that do not inherently have adenosine receptors. Examples of cells that do not have adenosine receptors include cells that do not have an adenosine receptor gene and cells that have an adenosine receptor gene but do not express the gene. Modifying cells so as not to have adenosine receptors can be achieved, for example, by knocking out (i.e., destroying) the adenosine receptor gene.

[0046] The term "adenosine receptor" refers to a G protein-coupled receptor (GPCR) that is responsive to adenosine. "Responsive to adenosine" may mean activated by adenosine. The adenosine receptor may be, for example, an adenosine receptor coupled to a Gs protein. Examples of adenosine receptors include adenosine receptors A1, A2A, A2B, and A3. Examples of adenosine receptors include, in particular, adenosine receptors A2A and A2B. The adenosine receptors A2A and A2B may be adenosine receptors coupled to a Gs protein. As the adenosine receptor, one adenosine receptor may be selected, or two or more adenosine receptors may be selected. Examples of adenosine receptors that may be selected include, for example, adenosine receptors A2A and / or A2B. Examples of adenosine receptors that may be selected include, in particular, adenosine receptors A2A and A2B. Genes encoding adenosine receptors are also referred to as "adenosine receptor genes." The nucleotide sequences of adenosine receptor genes and the amino acid sequences of adenosine receptors from various organisms can be obtained from public databases such as NCBI and Ensembl. For example, the amino acid sequence of human adenosine receptor A2A is registered with NCBI as NP_000666.2. For example, the amino acid sequence of mouse adenosine receptor A2A is registered with NCBI as NP_033760.2. For example, the amino acid sequence of human adenosine receptor A2B is registered with NCBI as NP_000667.1. For example, the amino acid sequence of mouse adenosine receptor A2B is registered with NCBI as NP_031439.2.

[0047] Below, a method for reducing the activity of a protein such as an adenosine receptor will be described.

[0048] "Decreased protein activity" means that the activity of the protein is reduced compared to unmodified cells. Specifically, "decreased protein activity" means that the activity of the protein per cell is reduced compared to unmodified cells. "Unmodified cells" as used herein refer to control cells that have not been modified to reduce the activity of a target protein, and specifically may refer to cells that normally express a target protein that functions normally. Examples of control cells include the host cells exemplified above. In one embodiment, the activity of the protein may be reduced compared to CHO cells such as CHO-DG44 and CHO-K1. In another embodiment, the activity of the protein may be reduced compared to HEK cells such as HEK293 and HEK293T. In another embodiment, the activity of the protein may be reduced compared to COS cells such as COS-1. Note that "decreased protein activity" also encompasses cases where the activity of the protein is completely eliminated. "Decreased protein activity" may more specifically mean a decrease in the number of molecules of the protein per cell and / or a decrease in the function of the protein per molecule compared to unmodified cells. In other words, the "activity" in "decreased protein activity" does not necessarily mean the catalytic activity of the protein, but may also mean the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. "Number of molecules of protein per cell" may mean the average number of molecules of the protein per cell. Note that "decreased number of molecules of protein per cell" also includes cases where the protein is completely absent. Furthermore, "decreased function per molecule of protein" also includes cases where the function per molecule of the protein is completely lost. The degree of decrease in protein activity is not particularly limited, as long as the desired effect is obtained (e.g., in the case of an adenosine receptor, as long as the adenosine receptor response to a test substance is reduced to the desired extent). The activity of the protein may be reduced to, for example, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, or 0% of that of unmodified cells.

[0049] Modifications that reduce the activity of a protein can be achieved, for example, by reducing the expression of the gene encoding the protein. "Reduced gene expression" means that the expression of the gene is reduced compared to unmodified cells. "Reduced gene expression" specifically means that the expression level of the gene per cell is reduced compared to unmodified cells. "Expression level of the gene per cell" may refer to the average expression level of the gene per cell. "Reduced gene expression" may more specifically mean a reduction in the transcription level (mRNA level) of the gene and / or a reduction in the translation level (protein level) of the gene. "Reduced gene expression" also includes cases where the gene is not expressed at all. "Reduced gene expression" is also referred to as "attenuated gene expression." Gene expression may be reduced to, for example, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, or 0% of that of unmodified cells.

[0050] Decreased gene expression may be due to, for example, decreased transcription efficiency, decreased translation efficiency, or a combination thereof. Decreased gene expression can be achieved, for example, by modifying the expression regulatory sequence of a gene. "Expression regulatory sequence" is a general term for a site that affects gene expression, such as a promoter. Expression regulatory sequences can be determined, for example, using a promoter search vector or genetic analysis software such as GENETYX. When modifying an expression regulatory sequence, preferably one or more bases, more preferably two or more bases, and particularly preferably three or more bases are modified in the expression regulatory sequence. Decreased gene transcription efficiency can be achieved, for example, by replacing the promoter of a gene on a chromosome with a weaker promoter. A "weaker promoter" refers to a promoter that weakens gene transcription compared to the native wild-type promoter. Examples of weaker promoters include inducible promoters. In other words, inducible promoters can function as weaker promoters under non-inducing conditions (e.g., in the absence of an inducer). Alternatively, a partial or entire region of the expression regulatory sequence may be deleted (deleted). In addition, the reduction of gene expression can also be achieved, for example, by introducing a mutation into the coding region of the gene that reduces gene expression. For example, the expression of the gene can be reduced by replacing the codons in the coding region of the gene with synonymous codons that are used less frequently in the host. In addition, the expression of the gene itself can be reduced, for example, by gene disruption as described below. In addition, the reduction of gene expression can be achieved, for example, by RNA interference.

[0051] Furthermore, a modification that reduces the activity of a protein can be achieved, for example, by disrupting the gene encoding the protein. "Disrupting a gene" means that the gene is modified so that it does not produce a protein that functions normally. "Not producing a protein that functions normally" includes cases where no protein is produced from the gene at all, and cases where the gene produces a protein with reduced or lost function per molecule (e.g., activity or properties).

[0052] Gene disruption can be achieved, for example, by deleting (deleting) the gene on a chromosome. "Gene deletion" refers to the deletion of part or all of the coding region of a gene. Furthermore, the entire gene may be deleted, including the sequences before and after the coding region of the gene on the chromosome. The sequences before and after the coding region of the gene may include, for example, a gene expression regulatory sequence. As long as a reduction in protein activity can be achieved, the region to be deleted may be any region, such as the N-terminal region (the region encoding the N-terminal side of the protein), an internal region, or a C-terminal region (the region encoding the C-terminal side of the protein). Generally, the longer the region to be deleted, the more reliably the gene can be inactivated. The region to be deleted may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the entire length of the coding region of the gene. Furthermore, it is preferable that the reading frames of the sequences before and after the region to be deleted do not match. Reading frame mismatches can result in frameshifts downstream of the region to be deleted.

[0053] Gene disruption can also be achieved by, for example, introducing an amino acid substitution (missense mutation) into the coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting one or two bases (frameshift mutation) (Journal of Biological Chemistry 272:8611-8617(1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515(1998), Journal of Biological Chemistry 26 116, 20833-20839(1991)).

[0054] Gene disruption can also be achieved, for example, by inserting another base sequence into the coding region of the gene on the chromosome. The insertion site may be anywhere in the gene, but the longer the inserted base sequence, the more reliably the gene can be inactivated. Furthermore, it is preferable that the reading frames of the sequences before and after the insertion site do not match. A mismatch in the reading frame can cause a frameshift downstream of the insertion site. The other base sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples include marker genes such as antibiotic resistance genes and genes useful for producing target substances.

[0055] Gene disruption may be carried out, particularly to delete (delete) the amino acid sequence of the encoded protein. In other words, modification that reduces the activity of a protein can be achieved, for example, by deleting the amino acid sequence of the protein (partial or entire region of the amino acid sequence), specifically by modifying the gene to encode a protein from which the amino acid sequence (partial or entire region of the amino acid sequence) has been deleted. The term "deletion of the amino acid sequence of a protein" refers to the deletion of part or entire region of the amino acid sequence of a protein. The term "deletion of the amino acid sequence of a protein" refers to the absence of the original amino acid sequence in the protein, and also encompasses cases in which the original amino acid sequence is changed to a different amino acid sequence. For example, a region that has been changed to a different amino acid sequence due to frameshifting may be considered a deleted region. While deletion of the amino acid sequence of a protein typically shortens the overall length of the protein, it may also be possible for the overall length of the protein to remain unchanged or to be extended. For example, deletion of part or entire region of the coding region of a gene can delete the region encoded by the deleted region in the amino acid sequence of the encoded protein. For example, by introducing a stop codon into the coding region of a gene, the region coded for by the region downstream of the introduction site in the amino acid sequence of the encoded protein can be deleted. For example, by causing a frameshift in the coding region of a gene, the region coded for by the frameshift site can be deleted. The position and length of the region to be deleted in the deletion of an amino acid sequence can be determined mutatis mutandis from the explanation of the position and length of the region to be deleted in the deletion of a gene.

[0056] The chromosome modification method can be selected appropriately depending on various conditions, such as the type of modification and the type of cell. For example, site-specific modification of the chromosome of higher eukaryotic cells, such as animal cells, can be performed using techniques utilizing various nucleases. Specific examples of such techniques include the CRISPR / Cas (CRISPR: clustered regularly interspaced short palindromic repeat, Cas: CRISPR-associated protein) method, the TALEN (transcription-activator like effector nuclease) method, and the ZFN (zinc finger nuclease) method. In these techniques, Cas, TALEN, and ZFN are used as nucleases, respectively. For example, a target site on a chromosome can be deleted by non-homologous end joining, or the target site on a chromosome can be replaced with another base sequence (such as a base sequence containing a marker gene) by homologous recombination. For example, a disrupted gene that has been modified so that it does not produce a normally functioning protein can be prepared, and donor DNA containing the disrupted gene can be introduced into a host cell to induce homologous recombination between the disrupted gene and the wild-type gene on the chromosome, thereby replacing the wild-type gene on the chromosome with the disrupted gene. Examples of disrupted genes include genes in which part or all of the coding region of a gene has been deleted, genes in which a missense mutation has been introduced, genes in which a nonsense mutation has been introduced, genes in which a frameshift mutation has been introduced, and genes in which an insertion sequence such as a transposon or a marker gene has been inserted.

[0057] Modifications that reduce the activity of a protein may also be performed by, for example, mutation treatments, such as X-ray irradiation, ultraviolet irradiation, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0058] The above-mentioned methods for reducing protein activity may be used alone or in any combination.

[0059] The decrease in the activity of the protein can be confirmed by measuring the activity of the protein. The activity of the adenosine receptor can be measured, for example, using the response of the adenosine receptor to adenosine as an index. The response of the adenosine receptor to adenosine can be measured, for example, by contacting adenosine with a cell and measuring the activation of the adenosine receptor. The activation of the adenosine receptor can be measured in the same manner as the activation of the olfactory receptor.

[0060] A decrease in protein activity can also be confirmed by confirming a decrease in expression of the gene encoding the protein. A decrease in gene expression can be confirmed by confirming a decrease in the transcription level of the gene or a decrease in the amount of protein expressed from the gene.

[0061] The reduction in the transcription level of a gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of unmodified cells. Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, microarray, RNA-Seq, etc. (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of mRNA may be reduced to, for example, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, or 0% of that of unmodified cells.

[0062] The reduction in the amount of protein can be confirmed by performing SDS-PAGE and checking the intensity of the separated protein bands. Alternatively, the reduction in the amount of protein can be confirmed by Western blotting using an antibody (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of protein (e.g., the number of molecules per cell) may be reduced to, for example, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, or 0% of that of unmodified cells.

[0063] Gene disruption can be confirmed by determining the nucleotide sequence, restriction enzyme map, or full length of a part or all of the gene, depending on the means used for disruption.

[0064] The above-mentioned methods for reducing protein activity can be used to reduce the activity of any protein or the expression of any gene.

[0065] Cells having an olfactory receptor gene can be used as cells having an olfactory receptor (cells of the present invention) either as is or after appropriate expression of the olfactory receptor gene. That is, if cells having an olfactory receptor gene already express the olfactory receptor gene, the cells may be used as cells having an olfactory receptor (cells of the present invention) as is. Alternatively, cells having an olfactory receptor (cells of the present invention) can be obtained by expressing the olfactory receptor gene in cells having an olfactory receptor gene. For example, the olfactory receptor gene can be expressed by culturing cells having an olfactory receptor gene, thereby obtaining cells having an olfactory receptor (cells of the present invention). Specifically, for example, after introduction (e.g., transfection) of the olfactory receptor gene, the host cells can be continuously cultured to express the olfactory receptor gene. The medium composition and culture conditions are not particularly limited as long as the cells having the olfactory receptor gene can be maintained (e.g., proliferated) and the olfactory receptor gene is expressed. During culture, cells having an olfactory receptor gene may or may not proliferate. The medium composition and culture conditions can be appropriately set depending on various conditions, such as the type of host cell. Culturing can be performed using, for example, a conventional medium and conditions used for culturing cells such as animal cells, either as is or with appropriate modifications. Specific examples of media that can be used for culturing animal cells include Opti-MEM medium (Thermo Fisher Scientific), DMEM medium, RPMI 1640 medium, and CD293 medium. Culturing can be performed, for example, at 36°C to 38°C under 5% CO ２ CO etc. ２ The culture can be carried out by static culture in a culture-containing atmosphere. If necessary, a selective drug or an expression inducer can be used.

[0066] Expression of olfactory receptors can be confirmed by measuring the response of the olfactory receptors to substances that induce a response of the olfactory receptors, or by measuring the amount of mRNA transcribed from the olfactory receptor gene or by detecting the olfactory receptors by Western blotting using antibodies.

[0067] The cells of the present invention can be used in the methods of the present invention, for example, as they are (as they are contained in the culture) or after being recovered from the medium. Furthermore, the culture or cells recovered therefrom may be used in the methods of the present invention after, for example, appropriate treatment such as washing, concentration, dilution, or fixation. Thus, the cells of the present invention may be used, for example, in a form isolated to a desired degree, or in a form contained in a material such as a culture.

[0068] Cell membranes can also be prepared from the cells of the present invention and used in the methods of the present invention. The cell membranes prepared from the cells of the present invention are cell membranes having olfactory receptors. Specifically, the cell membranes having olfactory receptors can be obtained, for example, as membrane fractions obtained when the cells of the present invention are disrupted. The cell membranes having olfactory receptors can be used, for example, as they are or dispersed in an artificial lipid bilayer membrane. The cell membranes having olfactory receptors can also be used in the form of vesicles (i.e., vesicles prepared from the cell membrane).

[0069] A cell membrane can be used, for example, to generate a space separated by the cell membrane. A cell membrane can be used, for example, to separate two spaces, such as two wells. That is, a cell membrane can be used to provide a reaction system having two spaces, such as two wells, separated from each other by a cell membrane. At least a portion of the boundary between such two spaces may be separated by the cell membrane. Such a reaction system may be provided, for example, as an ion channel measurement device (Kawano R. et al., Automated Parallel Recordings of Topologically Identified Single Ion Channels, Scientific Reports, 3, No. 1995 (2013)).

[0070] <3> Method of the Present Invention The method of the present invention can be carried out in vitro.

[0071] Step (A) is a step of contacting an olfactory receptor with a test substance. That is, first, the olfactory receptor and the test substance can be contacted.

[0072] A system in which contact between an olfactory receptor and a test substance is carried out is also referred to as a "reaction system." Contact between an olfactory receptor and a test substance can be carried out in an appropriate liquid. A liquid in which contact between an olfactory receptor and a test substance is carried out is also referred to as a "reaction solution." That is, for example, contact between an olfactory receptor and a test substance can be achieved by coexisting the olfactory receptor and the test substance in an appropriate reaction solution. Specifically, for example, contact between an olfactory receptor and a test substance can be achieved by dissolving, suspending, dispersing, or the like an olfactory receptor (e.g., a form such as one of the above-mentioned examples, such as a cell having an olfactory receptor) and a test substance in an appropriate liquid medium. Examples of the liquid medium include aqueous media such as water and aqueous buffer solutions. Note that when two or more components are contacted together with an olfactory receptor, contact between the components and the olfactory receptor may or may not begin simultaneously. That is, for example, after contact between a certain component and the olfactory receptor has begun, another component may be added to the reaction system. The reaction conditions (conditions for contacting the olfactory receptor with the test substance) are not particularly limited as long as the response of the olfactory receptor to the test substance can be measured. The reaction conditions can be appropriately set depending on various conditions, such as the form of use of the olfactory receptor, the type of test substance, and the method for measuring the response of the olfactory receptor. For example, known reaction conditions for measuring interactions between substances, such as interactions between proteins and ligands, may be used as is, or may be appropriately modified. The concentration of the test substance may be, for example, 0.01% (w / w) or more, 0.02% (w / w) or more, 0.05% (w / w) or more, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.5% (w / w) or more, or 1% (w / w) or more, or 15% (w / w) or less, 10% (w / w) or less, 7% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 0.5% (w / w) or less, 0.2% (w / w) or less, or 0.1% (w / w) or less, or a compatible combination thereof. Specifically, the concentration of the test substance may be, for example, 0.01 to 15% (w / w), 0.05 to 7% (w / w), or 0.1 to 5% (w / w).The concentration of the test substance may be, for example, 0.01 nM to 500 mM, 10 nM to 100 mM, 1 μM to 10 mM, or 3 μM to 1 mM. The concentration of the olfactory receptor may be, for example, 1 pg / mL to 10 mg / mL. Furthermore, when cells having olfactory receptors are used, the concentration of the cells having olfactory receptors may be, for example, 10 cell / mL to 10,000,000 cell / mL. The contact between the olfactory receptors and the test substance may or may not be terminated at an appropriate time point. The contact between the olfactory receptors and the test substance may generally be continued until the time of measuring the response of the olfactory receptor to the test substance. The duration of contact between the olfactory receptor and the test substance may be, for example, 0.1 second or more, 0.5 seconds or more, 1 second or more, 5 seconds or more, 10 seconds or more, 30 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, or 2 hours or more, or 24 hours or less, 12 hours or less, 6 hours or less, 2 hours or less, or 1 hour or less, or a compatible combination thereof. Specifically, the duration of contact between the olfactory receptor and the test substance may be, for example, 1 hour to 6 hours. The reaction system may contain other components in addition to the olfactory receptor (e.g., in the form exemplified above, such as cells having an olfactory receptor) and the test substance, as long as the response of the olfactory receptor to the test substance can be measured. The other components can be appropriately selected depending on various conditions, such as the form of use of the olfactory receptor, the type of test substance, and the method for measuring the response of the olfactory receptor. Examples of other components include salts such as calcium salts, carbon sources such as glucose, other medium components, and pH buffers.

[0073] Furthermore, when measuring the inactivation of olfactory receptors by a test substance, the contact between the olfactory receptor and the test substance can be carried out, for example, in the presence of a substance that activates the olfactory receptor (hereinafter also referred to as an "olfactory receptor activator"). The expressions "contacting the olfactory receptor with the test substance in the presence of an olfactory receptor activator," "contacting the olfactory receptor with an olfactory receptor activator in the presence of a test substance," and "contacting the olfactory receptor with an olfactory receptor activator and a test substance" may be used interchangeably. The description of the contact between the olfactory receptor and the test substance can also be applied mutatis mutandis to the contact between the olfactory receptor and the olfactory receptor activator. The contact between the olfactory receptor activator and the test substance and the olfactory receptor may or may not be initiated simultaneously. For example, the test substance and the olfactory receptor activator may be mixed in advance and then contacted with the olfactory receptor. Furthermore, for example, the olfactory receptor activator may be added to the reaction system after the contact between the test substance and the olfactory receptor has begun, or the test substance may be added to the reaction system after the contact between the olfactory receptor activator and the olfactory receptor has begun. The concentration of the olfactory receptor activator may be, for example, 0.01 nM to 500 mM, 10 nM to 100 mM, 1 μM to 10 mM, or 3 μM to 1 mM. When the contact between the olfactory receptor and the test substance is carried out in the presence of an olfactory receptor activator, the phrase "duration of contact between the olfactory receptor and the test substance" may be interpreted as "duration of contact between the olfactory receptor and the olfactory receptor activator and the test substance."

[0074] The olfactory receptor activator is not particularly limited as long as it can activate the olfactory receptor. The olfactory receptor activator may be composed of a single component (i.e., a pure substance) or a combination of two or more components (i.e., a mixture). When the olfactory receptor activator is a mixture, the number of components constituting the mixture and their composition ratios are not particularly limited. The olfactory receptor activator may be a known substance or a novel substance. The olfactory receptor activator may be a natural product or an artificial product. The olfactory receptor activator may or may not be known to be able to activate the olfactory receptor. As the olfactory receptor activator, for example, one capable of activating the olfactory receptor may be selected from the test substances exemplified above and used.

[0075] Step (A) is carried out under the following condition (X): (X) Conditions under which the response of adenosine receptors to a test substance is reduced.

[0076] The condition (X) may be achieved, for example, by the cell of the present invention having the following characteristic (X1) or (X2): (X1) the cell has been modified to reduce the activity of adenosine receptors; (X2) the cell does not inherently have adenosine receptors.

[0077] The features (X1) and (X2) are as described above.

[0078] The condition (X) may be achieved, for example, by performing the step (A) in the presence of an adenosine receptor antagonist. When the condition (X) is achieved by performing the step (A) in the presence of an adenosine receptor antagonist and the inactivation of an olfactory receptor by a test substance is measured, the contact of the olfactory receptor with the olfactory receptor activator is also performed in the presence of an adenosine receptor antagonist.

[0079] As adenosine receptor antagonist, the antagonist of selected adenosine receptor can be used.For example, as adenosine receptor A2A antagonist, caffeine, theophylline, istradefylline, CGS-15943, SCH-58261, SCH-442416, ZM-241385 can be included.As adenosine receptor A2A antagonist, in particular, CGS-15943 can be included.In addition, as adenosine receptor A2B antagonist, for example, caffeine, theophylline, CGS-15943, CVT-6883, MRS-1706, MRS-1754, MRE-2029-F20, PSB-603, PSB-0788, PSB-1115 can be included. Antagonists of the adenosine receptor A2B include, inter alia, CGS-15943.

[0080] The concentration of the adenosine receptor antagonist is not particularly limited, as long as it can reduce the adenosine receptor response to the test substance to a desired level.The adenosine receptor response to the test substance may be reduced to, for example, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, or 0% of that when the adenosine receptor antagonist is not used.The concentration of the adenosine receptor antagonist can be appropriately set according to various conditions, such as the type of adenosine receptor antagonist.The concentration of the adenosine receptor antagonist may be, for example, 0.05 μM or more, 0.1 μM or more, 0.2 μM or more, 0.5 μM or more, or 1 μM or more, or 1000 μM or less, 100 μM or less, 10 μM or less, or 5 μM or less, or a combination thereof. The concentration of the adenosine receptor antagonist may be specifically, for example, 0.05 to 1000 μM, 0.1 to 100 μM, or 0.2 to 10 μM.

[0081] The timing of supplying adenosine receptor antagonist to reaction system is not particularly limited, as long as it can reduce the response of adenosine receptor to test substance to desired extent.For example, adenosine receptor antagonist can be supplied to reaction system before test substance or simultaneously with test substance.In addition, when using olfactory receptor activator, for example, adenosine receptor antagonist can be supplied to reaction system before olfactory receptor activator or simultaneously with olfactory receptor activator.

[0082] Step (B) is a step of measuring the response of the olfactory receptor to the test substance. That is, the response of the olfactory receptor to the test substance can then be measured. The response of the olfactory receptor to a substance is also referred to as "the substance eliciting a response of the olfactory receptor."

[0083] The response of the olfactory receptor to the test substance includes activation or inactivation of the olfactory receptor by the test substance.

[0084] The timing for measuring the olfactory receptor response to a test substance is not particularly limited, as long as the olfactory receptor response to the test substance is measurable when the test substance is a substance that induces an olfactory receptor response. The timing for measuring the olfactory receptor response to a test substance can be appropriately set depending on various conditions, such as the form of use of the olfactory receptor, the type of test substance, and the method for measuring the olfactory receptor response. Specifically, the timing for measuring the olfactory receptor response to a test substance may be any appropriate time from the time when contact between the olfactory receptor and the test substance begins to the time when the olfactory receptor response to the test substance disappears. The timing for measuring the olfactory receptor response to a test substance may be, for example, the time when the maximum olfactory receptor response to the test substance is obtained (e.g., the time when the degree of activation or inactivation of the olfactory receptor by the test substance is maximum). Furthermore, the timing for measuring the olfactory receptor response to the test substance may be, for example, 0.1 seconds or later, 0.5 seconds or later, 1 second or later, 5 seconds or later, 10 seconds or later, 30 seconds or later, 1 minute or later, 5 minutes or later, 10 minutes or later, 30 minutes or later, 1 hour or later, or 2 hours or later from the time when contact between the olfactory receptor and the test substance begins, or up to 24 hours, 12 hours, 6 hours, 2 hours, or 1 hour, or any compatible combination thereof. Specifically, the timing for measuring the olfactory receptor response to the test substance may be, for example, from 1 hour to 6 hours from the time when contact between the olfactory receptor and the test substance begins. When contact between the olfactory receptor and the test substance is carried out in the presence of an olfactory receptor activator, the phrase "the time when contact between the olfactory receptor and the test substance begins" may be interpreted as "the time when contact between the olfactory receptor and the olfactory receptor activator and the test substance begins."

[0085] The activation or inactivation of the olfactory receptor by the test substance may be determined using as an index the degree of activation of the olfactory receptor (degree of activation D1) when the step (A) is carried out (i.e., under conditions in which the olfactory receptor is brought into contact with the test substance). That is, the step (B) may be, for example, (B1) a step of measuring the degree of activation D1.

[0086] Specifically, the activation or inactivation of the olfactory receptor by the test substance may be determined by comparing the degree of activation of the olfactory receptor (degree of activation D1) when step (A) is carried out (i.e., under conditions in which the olfactory receptor is brought into contact with the test substance) with the degree of activation of the olfactory receptor under control conditions (degree of activation D2).

[0087] "Control conditions" refer to the following conditions (C2-1) or (C2-2): (C2-1) conditions in which the olfactory receptor is not contacted with the test substance; (C2-2) conditions in which the olfactory receptor is contacted with the test substance, and the concentration of the test substance is lower than the concentration of the test substance in step (A).

[0088] When the contact between the olfactory receptor and the test substance is carried out in the presence of an olfactory receptor activator, the above condition (C2-1) or (C2-2) can be interpreted as follows: (C2-1) A condition in which the olfactory receptor is contacted with the olfactory receptor activator, but not with the test substance (i.e., a condition in which the olfactory receptor is contacted with the olfactory receptor activator in the absence of the test substance); (C2-2) A condition in which the olfactory receptor is contacted with the test substance in the presence of an olfactory receptor activator, wherein the concentration of the test substance is lower than the concentration of the test substance in step (A).

[0089] In other words, activation or inactivation of olfactory receptors by a test substance may be determined, for example, using as an index the difference in the degree of activation of olfactory receptors due to the presence or absence or different concentrations of the test substance.

[0090] The above condition (C2-1) includes conditions before contacting the olfactory receptor with the test substance. The above condition (C2-1) also includes conditions after contacting the olfactory receptor with the test substance, in which the test substance is substantially (e.g., completely) removed from the reaction system and the olfactory receptor response to the test substance is substantially (e.g., completely) eliminated. The concentration of the test substance under the above condition (C2-2) is not particularly limited, as long as a measurable difference is observed between the degree of activation D1 and the degree of activation D2. The concentration of the test substance under the above condition (C2-2) may be, for example, 90% or less, 70% or less, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the concentration of the test substance in step (A). Other than the presence or absence or concentration of the test substance, the control condition is not particularly limited, as long as it allows the response of the olfactory receptor to the test substance to be evaluated. The control conditions may be the same as the conditions of step (A), except for the presence or absence or concentration of the test substance. For example, if condition (X) is achieved by performing step (A) in the presence of an adenosine receptor antagonist, the contact between the olfactory receptor and the substance in the control conditions is also performed in the presence of the adenosine receptor antagonist.

[0091] The method of the present invention may include a step of measuring the degree of activation D2. The degree of activation D1 and the degree of activation D2 may be measured in a single reaction system with a time lag, or may be measured simultaneously or with a time lag in separate reaction systems. The degree of activation D2 may be measured before or after the degree of activation D1. For example, after measuring the degree of activation D2, a test substance may be added to the reaction system and the degree of activation D1 may be measured.

[0092] When the degree of activation D1 is high, it may be determined that the test substance has activated the olfactory receptor. Specifically, when the degree of activation D1 is higher than the degree of activation D2, it may be determined that the test substance has activated the olfactory receptor. For example, when the ratio of the degree of activation D1 to the degree of activation D2 (i.e., D1 / D2) is 1.5 or more, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, or 100 or more, it may be determined that the test substance has activated the olfactory receptor. Examples of the ratio of the degree of activation D1 to the degree of activation D2 include the normalized response values ​​described in the Examples.

[0093] When the degree of activation D1 is low, it may be determined that the inactivation of the olfactory receptor by the test substance has been observed. Specifically, when the degree of activation D1 is lower than the degree of activation D2, it may be determined that the inactivation of the olfactory receptor by the test substance has been observed. For example, when the ratio of the degree of activation D1 to the degree of activation D2 in the presence of an olfactory receptor activator (i.e., D1 / D2) is 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less, it may be determined that the inactivation of the olfactory receptor by the test substance has been observed. Examples of the ratio of the degree of activation D1 to the degree of activation D2 in the presence of an olfactory receptor activator include the normalized response values ​​described in the Examples obtained in the presence of an olfactory receptor activator.

[0094] The method for measuring the response of an olfactory receptor to a test substance is not particularly limited. The method for measuring the response of an olfactory receptor to a test substance can be appropriately selected depending on various conditions, such as the form of use of the olfactory receptor and the type of response to be measured. That is, the response of an olfactory receptor to a test substance can be measured, for example, by an appropriate method that can measure the activation or inactivation of the olfactory receptor by the test substance.

[0095] The method for measuring the activation or inactivation of olfactory receptors by a test substance is not particularly limited. The activation or inactivation of olfactory receptors by a test substance can be measured, for example, by known methods for measuring the activity of receptors such as olfactory receptors. Examples of such methods include methods for measuring intracellular calcium concentration and intracellular cAMP concentration. That is, the activation or inactivation of olfactory receptors by a test substance can be measured, for example, using intracellular calcium concentration or intracellular cAMP concentration as an indicator. Specifically, the activation or inactivation of olfactory receptors by a test substance can be measured, for example, using cells having olfactory receptors, using intracellular calcium concentration or intracellular cAMP concentration as an indicator. The activation or inactivation of olfactory receptors by a test substance can be measured, in particular, using intracellular cAMP concentration as an indicator. For example, in HEK293T cells, when olfactory receptors are activated by odorants, they couple with intracellular G proteins (specifically, Gs, such as Golf) to activate adenylate cyclase, thereby increasing the amount of intracellular cAMP (Kajiya K. et al., Molecular bases of odor discrimination: Reconstitution of olfactory receptors that recognize overlapping sets of odorants. Journal of Neuroscience, 2001, 21:6018-6025). Techniques for measuring intracellular cAMP levels include, for example, ELISA and reporter assays. An example of a reporter assay is luciferase assay. Reporter assays can measure intracellular cAMP levels using a reporter gene (e.g., luciferase gene) whose expression is dependent on cAMP levels. An example of a technique for measuring intracellular calcium levels is calcium imaging. Calcium imaging can measure intracellular calcium levels using calcium indicators. Examples of calcium indicators include calcium-sensitive fluorescent dyes and calcium-sensitive fluorescent proteins.Examples of calcium-sensitive fluorescent dyes include Fura 2 and Fluo 4. Examples of calcium-sensitive fluorescent proteins include Cameleon, TN-XL, GCaMP, and G-GECO. "Calcium concentration" may refer to the concentration of free calcium ions.

[0096] The description of the measurement of olfactory receptor activation or inactivation using cells having olfactory receptors can also be applied mutatis mutandis to the case where a cell membrane having olfactory receptors is used, particularly when the cell membrane having olfactory receptors is used in a form having an internal space.

[0097] For example, a cell membrane having an olfactory receptor can be used to generate a space separated by the cell membrane, and the activation or inactivation of the olfactory receptor can be measured in the same manner as when using a cell having an olfactory receptor.Specifically, for example, when a cell membrane is used to separate two spaces, that is, when a cell membrane is used to provide a reaction system with two spaces, and the two spaces are separated from each other by the cell membrane, the activation or inactivation of the olfactory receptor can be measured in the same manner as when using a cell having an olfactory receptor.In such a case, the space separated by the cell membrane can be considered the inside of the cell (also referred to as "internal space").Specifically, one of the two spaces can be considered the inside of the cell (also referred to as "internal space"), and the other can be considered the outside of the cell (also referred to as "external space").Of these spaces, the one containing the test substance can be considered the external space. In such cases, the terms "intracellular calcium concentration" and "intracellular cAMP concentration" in the description of measuring olfactory receptor activation or inactivation using cells having olfactory receptors can be read as "calcium concentration within the internal space" and "cAMP concentration within the internal space," respectively.

[0098] In either case, measurable parameters can be selected depending on the manner in which the olfactory receptor is used.

[0099] Note that "measuring a certain parameter and using it as an index for measuring the response of an olfactory receptor to a test substance" means that as long as the response can be measured (i.e., it can be determined whether the response is observed), it is sufficient to obtain and use data reflecting the parameter, and it is not necessary to obtain the value of the parameter itself. In other words, when data reflecting a certain parameter is obtained, it is not necessary to calculate the value of the parameter itself from the data. Specifically, for example, when measuring intracellular cAMP concentration by luciferase assay and using the data as an index for measuring the activation or inactivation of an olfactory receptor by a test substance, it is sufficient to obtain and use data reflecting the intracellular cAMP concentration (e.g., luminescence intensity) as long as the activation or inactivation can be measured (i.e., it can be determined whether the activation or inactivation is observed), and it is not necessary to calculate the intracellular cAMP concentration itself from the data.

[0100] The purpose of measuring the response of the olfactory receptor to a test substance is not particularly limited. The measurement results of the response of the olfactory receptor to a test substance can be used, for example, to evaluate the properties of the test substance (e.g., properties that can be associated with the olfactory receptor, such as the property of exhibiting or suppressing a specific odor). That is, for example, if the selected olfactory receptor is an olfactory receptor that is activated by a substance that exhibits a specific odor, the test substance may be evaluated as being capable of exhibiting the odor when the olfactory receptor is activated by the test substance, or may be evaluated as being capable of suppressing the odor when the olfactory receptor is inactivated by the test substance.

[0101] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0102] <1> Preparation of cells expressing human olfactory receptors <1-1> Preparation of expression vectors for human olfactory receptors 352 types of human olfactory receptors (OR1A1, OR1A2, OR1B1, OR1C1, OR1D2, OR1D5, OR1E1, OR1F1, OR1F12, OR1G1, OR1I1, OR1J1, OR1J2, OR1J4, OR1K1, OR1L1, OR1L3, OR1L4, OR1L8, OR1M1, OR1N1, OR1N2, OR1Q1, OR1R1P, OR1S1, OR2A1, OR2A2, OR2A4, OR2A5, OR2A12, OR2A14, OR2A25, OR2AE1, OR2AG1, OR2 AG2, OR2AJ1P, OR2AK2, OR2AP1, OR2AT4, OR2B2, OR2B3, OR2B6, OR2B11, OR2C1, OR2C3, OR2D2, OR2D3, OR2F1, OR2G2, OR2G3, OR2G6, OR2H1, OR2H2, O R2J2, OR2J3, OR2K2, OR2L2, OR2L8, OR2L13, OR2M2, OR2M4, OR2M7, OR2S2, OR2T1, OR2T2, OR2T5, OR2T6, OR2T8, OR2T10, OR2T11, OR2T27, OR2T34, OR 2V2, OR2W1, OR2W3, OR2Y1, OR2Z1, OR3A1, OR3A2, OR3A3, OR3A4, OR4A5, OR4A15, OR4A16, OR4A47, OR4B1, OR4C3, OR4C5, OR4C6, OR4C11, OR4C12, OR 4C13, OR4C15, OR4C16, OR4C46, OR4D1, OR4D2, OR4D5, OR4D6, OR4D9, OR4D10, OR4D11, OR4E2, OR4F3, OR4F5, OR4F6, OR4F14P, OR4F15, OR4G11P, OR4 H12P, OR4K1, OR4K2, OR4K5, OR4K13, OR4K14, OR4K15, OR4K17, OR4L1, OR4M1, OR4N2, OR4N4, OR4N5, OR4P4, OR4Q3, OR4S1, OR4S2, OR4X1, OR4X2, OR 5A1, OR5A2, OR5AC2, OR5AK2, OR5AK3P, OR5AN1, OR5AP2, OR5AR1, OR5AS1, OR5AU1, OR5B2, OR5B3, OR5B12, OR5B17, OR5B21, OR5C1, OR5D13, OR5D14,OR5D16、OR5D18、OR5F1、OR5H1、OR5H2、OR5H6、OR5H14、OR5I1、OR5J2、OR5K1、OR5K3、OR5K4、OR5L2、OR5M3、OR5M8、OR5M9、OR5M10、OR5M11、OR5P3、OR5R1、OR5T1、OR5T2、OR5T3、OR5V1、OR5W2、OR6A2、OR6B1、OR6B2、OR6C1、OR6C2、OR6C3、OR6C4、OR6C6、OR6C65、OR6C66P、OR6C68、OR6C70、OR6C74、OR6C75、OR6C76、OR6F1、OR6J1、OR6K2、OR6K3、OR6K6、OR6M1、OR6N1、OR6N2、OR6P1、OR6Q1、OR6S1、OR6T1、OR6V1、OR6X1、OR6Y1、OR7A3P、OR7A5、OR7A10、OR7A17、OR7C1、OR7C2、OR7D2、OR7D4、OR7E24、OR7G1、OR7G2、OR7G3、OR8A1、OR8B3、OR8B4、OR8B8、OR8B12、OR8D1、OR8D2、OR8D4、OR8G2、OR8G5、OR8H3、OR8I2、OR8J1、OR8J3、OR8K1、OR8K3、OR8K5、OR8S1、OR8U1、OR9A4、OR9G1、OR9G4、OR9I1、OR9K2、OR9Q1、OR9Q2、OR10A3、OR10A4、OR10A5、OR10A6、OR10A7、OR10AD1、OR10AG1、OR10C1、OR10D3、OR10D4P、OR10G2、OR10G3、OR10G4、OR10G6、OR10G7、OR10G9、OR10H2、OR10H4、OR10J1、OR10J3、OR10J5、OR10K1、OR10K2、OR10P1、OR10Q1、OR10R2、OR10S1、OR10T2、OR10V1、OR10W1、OR10X1、OR10Z1、OR11A1、OR11G2、OR11H4、OR11H6、OR11H12、OR11L1、OR12D2、OR12D3、OR13A1、OR13C2、OR13C3、OR13C4、OR13C8、OR13D1、OR13F1、OR13G1、OR13H1、OR13J1、OR14A2、OR14A16、OR14C36、OR14I1、OR14J1、OR14K1、OR14L1P、OR51A1P、OR51A4、OR51A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51D1, OR51E1, OR51E2, OR51F1, OR51F2, OR51F5P, OR51G1, OR51G2, OR51H 1, OR51I1, OR51I2, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52 B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E8, OR52H1, OR52I2, OR52J3, OR52K2, OR52L2P, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52P2P, OR52R1, OR52W1, OR52Z1P, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B2P, OR56B4).

[0103] We purchased 352 human olfactory receptor genes from the TrueClone cDNA Clone Collection (OriGene). Using primers designed based on the sequences registered in GenBank, we amplified subcloning fragments for each of the 352 human olfactory receptor genes by PCR using the purchased human olfactory receptor genes as templates. The amplified subcloning fragments for each gene were subcloned downstream of the Rho tag sequence in the Rho-pME18S vector (K. Kajiya et al., Journal of Neuroscience, 15 August 2001, 21 (16) 6018-6025) using the EcoRI and XhoI sites, yielding 352 expression vectors for human olfactory receptors.

[0104] <1-2> Preparation of Olfactory Receptor-Expressing Cells HEK293T cells expressing each of the 352 olfactory receptors were prepared using the following procedure. The gene mixture shown in Table 1 and the transfection reagent mixture shown in Table 2 were prepared and left to stand at room temperature for 5 minutes. pcDNA3.1-microbat RTP1s is an expression vector for bat RTP1s, pcDNA3.1-Golf is an expression vector for human Golf, and pcDNA3.1-Ric8B is an expression vector for rat Ric8B (JP Patent Publication No. 2019-037197). The gene mixture and transfection reagent mixture were mixed and dispensed in 12.5 μL aliquots into each well of a poly-D-lysine-coated 384-well plate. The mixture was left to stand for 15 minutes in a clean bench. HEK293T cells (2.5 x 10 cells) were seeded in 10 cm dishes the day before. 6 1.2 x 10 cells / 10 cm dish 5 The solution was adjusted to a concentration of 1000 cells / mL, and 25 μL of the solution was seeded into each well of a 384-well plate. The cells were then cultured overnight in an incubator maintained at 37°C and 5% CO2. In this manner, 352 HEK293T cell cultures transfected with the expression vectors shown in Table 1 and expressing the genes encoded by those expression vectors were obtained. As a control, the human olfactory receptor expression vector in the gene mixture shown in Table 1 was replaced with the empty vector Rho-pME18S, and the same procedure was repeated to obtain a culture of HEK293T cells transfected with the empty vector Rho-pME18S (hereinafter also referred to as "control cells").

[0105]

[0106]

[0107] <2> Measurement of olfactory receptor response to test substances <2-1> Luciferase assay Olfactory receptor-expressing cells were used to measure the olfactory receptor response to test substances. The test substances used were Japanese-style dashi (product name: Hon-Dashi® Dried Sardine Dashi), soy sauce, noodle soup, chicken stock (product name: Marutori Gara Soup®), oyster sauce, beef extract, powdered coffee, and corn soup (product name: Knorr® Corn Cream).

[0108] The 352 olfactory receptors expressed in HEK293T cells conjugate with Golf to activate adenylate cyclase, thereby increasing intracellular cAMP levels. In this example, a luciferase reporter gene assay was used to measure the response of olfactory receptors to test substances. This assay monitors the increase in intracellular cAMP levels as an increase in luminescence intensity derived from firefly luciferase. The "luciferase reporter gene assay" is also referred to as the "luciferase assay." Firefly luciferase is expressed from the firefly luciferase gene carried by the pGL4.29[luc2P / CRE / Hygro] Vector in a manner dependent on the amount of intracellular cAMP. Additionally, the luminescence intensity derived from Renilla luciferase was used as an internal standard to correct for errors in gene transfer efficiency and cell number in each well. Renilla luciferase is constitutively expressed from the Renilla luciferase gene carried in the pGL4.74[hRluc / TK] Vector under the control of the TK promoter.

[0109] The medium was removed from the cultures obtained in <1-2> above, and 60 μL of test substance solution was added to each to obtain a reaction solution. Each test substance solution was prepared by dissolving the test substance in CD293 (Life Technologies, Inc.). When an adenosine receptor antagonist (described below) was used, the adenosine receptor antagonist was also dissolved in each test substance solution. The reaction solution was placed in an incubator maintained at 37°C and 5% CO2, and the cells were cultured for 4 hours to allow sufficient intracellular expression of the firefly luciferase gene. The luminescence value derived from intracellular firefly luciferase was measured and designated the "Luc value." In addition, the luminescence value derived from intracellular Renilla luciferase was measured and designated the "hRLuc value." The luminescence value derived from each luciferase was measured using Dual-Glo TM Measurement was performed using a luciferase assay system (Promega) according to the product's operating manual.

[0110] <2-2> Calculation of olfactory receptor activity The luminescence value (Luc value) derived from firefly luciferase induced by stimulation with the test substance was divided by the luminescence value (hRluc value) derived from Renilla luciferase in the same well to obtain the "Luc / hRluc value." The Luc / hRluc value in cells stimulated with the test substance was divided by the Luc / hRluc value in cells not stimulated with the test substance to obtain the "fold increase." Furthermore, the fold increase in cells transfected with the olfactory receptor expression vector was divided by the fold increase in control cells (cells transfected with the empty vector Rho-pME18S) to obtain the "normalized response." The common logarithm of the normalized response was used as the "olfactory receptor activity," a quantitative index of the response strength of the olfactory receptor to the test substance.

[0111] <3> Cellular response to adenosine and inhibition of response by adenosine receptor antagonists. Luciferase assays were performed on control cells (cells transfected with the empty vector Rho-pME18S) using the same procedures as in <2-1> to <2-2>, except that adenosine was used instead of the test substance. The Luc / hRluc value was calculated for cells stimulated with adenosine. Separately, the Luc / hRluc value was calculated for cells stimulated with 20 μM adenosine in the presence of an adenosine receptor antagonist. The adenosine receptor antagonist used was CGS-15943, an antagonist of adenosine receptors A2A and A2B.

[0112] The results are shown in Figures 1 and 2. Control cells responded to adenosine (Figure 1), i.e., adenosine induced an increase in cAMP in control cells. The response to adenosine (i.e., the increase in cAMP due to adenosine) was suppressed by an adenosine receptor antagonist (Figure 2).

[0113] <4> Non-specific cellular responses to test substances and inhibition of non-specific responses by adenosine receptor antagonists For control cells (cells transfected with the empty vector Rho-pME18S) and olfactory receptor-expressing cells, the non-specific cellular responses to the test substances (i.e., responses other than the olfactory receptor response to the test substance) were evaluated based on the Luc / hRluc value when stimulation with the test substance was performed in the presence or absence of 10 μM adenosine receptor antagonist CGS-15943.

[0114] The results for control cells are shown in Figure 3. The control cells responded to several test substances, but the responses were almost completely inhibited by an adenosine receptor antagonist. Figure 4 shows the results for olfactory receptor-expressing cells, which express the olfactory receptor OR10G7, which responds to eugenol. The OR10G7-expressing cells responded to several test substances, but the responses were partially or almost completely inhibited by an adenosine receptor antagonist. These results demonstrate that adenosine receptors (specifically, adenosine receptors A2A and / or A2B) respond to test substances, which can be an obstacle when measuring olfactory receptor responses to test substances, and that adenosine receptor responses (i.e., nonspecific responses) can be inhibited by an adenosine receptor antagonist.

[0115] <5> Correlation between Adenosine Concentration in Test Substance and Nonspecific Cellular Response to Test Substance The adenosine concentration in the test substance is shown in Table 3. The correlation between the adenosine concentration during olfactory receptor activity measurement and the Luc value of olfactory receptor OR10G7-expressing cells is shown in Figure 5. The higher the adenosine concentration during olfactory receptor activity measurement, the higher the Luc value obtained, i.e., the stronger the response of OR10G7-expressing cells. This suggests that there is a positive correlation between the adenosine concentration in the test substance (specifically, the adenosine concentration during olfactory receptor activity measurement) and the nonspecific cell response to the test substance.

[0116]

[0117] <6> Improving the detection sensitivity of olfactory receptor responses to test substances using an adenosine receptor antagonist Figure 6 shows olfactory receptor activity (common logarithm of normalized response) measured in the presence or absence of 20 μM adenosine receptor antagonist CGS-15943 using Hon-Dashi® dried sardine stock (dissolved in DMSO to a concentration of 1.5%) as the test substance. Figure 7 shows olfactory receptor activity (common logarithm of normalized response) measured in the presence or absence of 20 μM adenosine receptor antagonist CGS-15943 using Knorr® Tsubu-Tappuri Corn Cream (dissolved in DMSO to a concentration of 3%) as the test substance. For both test substances, the use of an adenosine receptor antagonist increased the number of olfactory receptors that showed a strong response (activation or inactivation) to the test substance. In other words, it was revealed that adenosine receptor antagonists suppress non-specific responses of cells to test substances, thereby improving the detection sensitivity of the response (i.e., activation or inactivation) of olfactory receptors to test substances.

[0118] According to the present invention, the response of olfactory receptors to substances can be specifically measured.

Claims

Claim 1 A method for measuring the response of an olfactory receptor to a test substance, comprising: the following steps (A) and (B): (A) contacting the olfactory receptor with the test substance; and (B) measuring the response of the olfactory receptor to the test substance ; wherein the olfactory receptor is used in a form carried by a cell or a cell membrane prepared from the cell, and step (A) is carried out under the following condition (X): (X) a condition in which the response of the adenosine receptor to the test substance is reduced. Claim 2 The method according to claim 1, wherein the adenosine receptor is adenosine receptor A2A and / or A2B. Claim 3 The method according to claim 1, wherein condition (X) is achieved by carrying out step (A) in the presence of an adenosine receptor antagonist. Claim 4 The adenosine receptor antagonist is selected from the group consisting of caffeine, theophylline, istradefylline, CGS-15943, SCH-58261, SCH-442416, ZM-241385, CVT-6883, MRS-1706, MRS-1754 , MRE-2029-F20, PSB-603, PSB-0788, and PSB-1115, according to claim 3. Claim 5 The method according to claim 3, wherein the adenosine receptor antagonist is used at a concentration of 0.05 to 1000 μM. Claim 6 The method according to claim 1, wherein condition (X) is achieved by the cell having the following feature (X1) or (X2): (X1) the cell is modified such that the activity of the adenosine receptor is reduced; (X2) the cell does not inherently have the adenosine receptor. Claim 7 The method according to claim 6, wherein the cell is modified to not have the adenosine receptor. Claim 8 The method according to claim 1, wherein the response of the olfactory receptor is activation of the olfactory receptor. Claim 9 The method according to claim 1, wherein the response of the olfactory receptor is inactivation of the olfactory receptor, and step (A) is carried out in the presence of a substance that activates the olfactory receptor. Claim 10 The method according to claim 1, wherein the olfactory receptor is used in a form carried by the cell. Claim 11 The method according to claim 1, wherein the cell is an animal cell. Claim 12 The method according to claim 1, wherein the response is measured using the intracellular cAMP concentration or the intracellular calcium concentration as an indicator.

13. The method according to claim 12, wherein the intracellular cAMP concentration is measured by a reporter assay.

14. The method according to claim 1, wherein the test substance is selected from the group consisting of foods, their raw materials, and their fractions.

15. The olfactory receptor is OR1A1, OR1A2, OR1B1, OR1C1, OR1D2, OR1D5, OR1E1, OR1F1, OR1F12, OR1G1, OR1I1, OR1J1, OR1J2, OR1J4, OR1K1, OR1L1, OR1L3, OR1L4, OR1L8, OR1M1 , OR1N1, OR1N2, OR1Q1, OR1R1P, OR1S1, OR2A1, OR2A2, OR2A4, OR2A5, OR2A12, OR2A14, OR2A25, OR2AE1, OR2AG1, OR2AG2, OR2AJ1P, OR2AK2, OR2AP1, OR2AT4, OR2B2, OR2B3 , OR2B6, OR2B11, OR2C1, OR2C3, OR2D2, OR2D3, OR2F1, OR2G2, OR2G3, OR2G6, OR2H1, OR2H2, OR2J2, OR2J3, OR2K2, OR2L2, OR2L8, OR2L13, OR2M2, OR2M4, OR2M7, OR2S2, OR2T1, OR2T2, OR2T5, OR2T6, OR2T8, OR2T10, OR2T11, OR2T27, OR2T34, OR2V2, OR2W1, OR2W3, OR2Y1, OR2Z1, OR3A1, OR3A2, OR3A3, OR3A4, OR4A5, OR4A15, OR4A16, OR4A47, OR4B1, OR4C3, OR4C5, OR4C6, OR4C11, OR4C12, OR4C13, OR4C15, OR4C16, OR4C46, OR4D1, OR4D2, OR4D5, OR4D6, OR4D9, OR4D10, OR4D11, OR4E2, OR4F3, OR4F5, OR4F6, OR4F14P, OR4F15, OR4G11P, OR4H12P, OR4K1, OR4K2, OR4K5, OR4K13, OR4K14, OR4K15, OR4K17, OR4L1, OR4M1, OR4N2, OR4N4, OR4N5, OR4P4, OR4Q3, OR4S1, OR4S2, OR4X1, OR4X2, OR5A1, OR5A2, OR5AC2, OR5AK2, OR5AK3P, OR5AN1, OR5AP2, OR5AR1, OR5AS1, OR5AU1, OR5B2, OR5B3, OR5B12, OR5B17, OR5B21, OR5C1, OR5D13, OR5D14, OR5D16, OR5D18, OR5F1, OR5H1, OR5H2, OR5H6, OR5H14, OR5I1, OR5J2, OR5K1, OR5K3, OR5K4, OR5L2, OR5M3, OR5M8, OR5M9, OR5M10, OR5M11, OR5P3, OR5R1, OR5T1, OR5T2, OR5T3, OR5V1, OR5W2, OR6A2, OR6B1, OR6B2, OR6C1, OR6C2, OR6C3, OR6C4, OR6C6, OR6C65, OR6C66P, OR6C68, OR6C70, OR6C74, OR6C75, OR6C76, OR6F1, OR6J1, OR6K2, OR6K3, OR6K6, OR6M1, OR6N1 , OR6N2, OR6P1, OR6Q1, OR6S1, OR6T1, OR6V1, OR6X1, OR6Y1, OR7A3P, OR7A5, OR7A10 , OR7A17, OR7C1, OR7C2, OR7D2, OR7D4, OR7E24, OR7G1, OR7G2, OR7G3, OR8A1, OR8B3 , OR8B4, OR8B8, OR8B12, OR8D1, OR8D2, OR8D4, OR8G2, OR8G5, OR8H3, OR8I2, OR8J1, OR8J3, OR8K1, OR8K3, OR8K5, OR8S1, OR8U1, OR9A4, OR9G1, OR9G4, OR9I1, OR9K2, OR9Q1, OR9Q2, OR10A3, OR10A4, OR10A5, OR10A6, OR10A7, OR10AD1, OR10AG1, OR10C1, OR10D3, OR10D4P, OR10G2, OR10G3, OR10G4, OR10G6, OR10G7, OR10G9, OR10H2, OR10H4, OR10J1, OR10J3, OR10J5, OR10K1, OR10K2, OR10P1, OR10Q1, OR10R2, OR10S1, OR10T2, OR10V1, OR10W1, OR10X1, OR10Z1, OR11A1, OR11G2, OR11H4, OR11H6, OR11H12, OR11L1, OR12D2, OR12D3, OR13A1, OR13C2, OR13C3, OR13C4, OR13C8, OR13D1, OR13F1, OR13G1, OR13H1, OR13J1, OR14A2, OR14A16, OR14C36, OR14I1, OR14J1, OR14K1, OR14L1P, OR51A1P, OR51A4, OR51A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51D1, OR51E1, OR51E2, OR51F1, OR51F2, OR51F5P, OR51G1, OR51G2, OR51H1, OR51I1, OR51I2, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52 The method according to claim 1, selected from the group consisting of D1, OR52E2, OR52E4, OR52E5, OR52E8, OR52H1, OR52I2, OR52J3, OR52K2, OR52L2P, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52P2P, OR52R1, OR52W1, OR52Z1P, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B2P, and OR56B4.

16. The method according to claim 1, wherein the olfactory receptor is a human olfactory receptor.