Substrate for cells

Abstract

To provide a substrate for cells, which has excellent capability for detecting a chemical substance such as a smell substance.SOLUTION: A substrate for cells includes a compartment including a polypeptide-coated surface and is used for measuring the activity of a sensor protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cell substrate and the like. [Background technology]

[0002] Groups of odorants that characterize certain human diseases and mental states have been identified, and because they are highly useful as test markers, the development of various odor sensors targeting these has become active. Since the olfactory receptors of living organisms have excellent characteristics in terms of diversity, sensitivity, selectivity, etc. that are not found in conventional odor sensor elements such as semiconductors, the development of new odor sensors that use olfactory receptors as sensor elements is expected.

[0003] Patent Document 1 discloses the use of cells expressing modified olfactory receptors or lipid bilayer membranes comprising modified olfactory receptors as odor sensors. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2022 / 024902 Summary of the Invention [Problem to be solved by the invention]

[0005] Since the production efficiency of odor sensors that have a process of artificially preparing a lipid bilayer membrane equipped with a sensor protein such as an olfactory receptor is not necessarily sufficient, there is a demand for further improvement in the production efficiency of odor sensors. Therefore, the inventor focused on using cells that express sensor proteins. In the course of conducting research based on this, the inventor found that there is a difference in the detectability of chemical substances such as odorants depending on the type of cell substrate.

[0006] Therefore, an object of the present disclosure is to provide a cell substrate that has excellent detectability for chemical substances such as odorants. [Means for solving the problem]

[0007] In view of the above problems, the present inventors have conducted intensive research and have found that the above problems can be solved by a cell substrate used for measuring the activity of a sensor protein, the cell substrate including a compartment including a polypeptide-coated surface. That is, the present disclosure includes the following aspects. That is, the present invention includes the following aspects.

[0008] Item 1. A cell substrate for use in measuring the activity of a sensor protein, comprising a compartment comprising a polypeptide-coated surface.

[0009] Item 2. The cell substrate according to Item 1, wherein the polypeptide is an extracellular matrix protein or a partial peptide thereof.

[0010] Item 3. The cell-substrate according to Item 1, wherein the polypeptide is collagen.

[0011] Item 4. The coating amount of the polypeptide is 0.5 μg / cm 2 Item 2. The cell substrate according to Item 1, which is as described above.

[0012] Item 5. The area of ​​the section is 1 to 50 mm 2 Item 2. The cell substrate according to Item 1,

[0013] Item 6. The cell substrate according to any one of Items 1 to 5, wherein the cells contain a polynucleotide that includes a coding sequence for a sensor protein.

[0014] Item 7. The cell substrate according to Item 6, wherein the sensor protein is an olfactory receptor protein.

[0015] Item 8. The cell substrate according to any one of Items 1 to 5, which is used to measure the activity of the sensor protein after replacing or adding a solution with cells disposed on the surface.

[0016] Item 9. The cell substrate according to any one of Items 1 to 5, wherein the cells are insect cells.

[0017] Item 10. A method for measuring the activity of a sensor protein, comprising measuring the activity of the sensor protein in cells placed on a polypeptide-coated surface. Effect of the Invention

[0018] According to the present disclosure, it is possible to provide a cell substrate that has excellent detectability for chemical substances such as odorants. [Brief description of the drawings]

[0019] [Figure 1] This shows an image of cells observed immediately after the plate was left standing overnight at 27° C. after seeding in Test Example 2. The gelatin concentration in the medium is shown on the left side of the photograph. [Diagram 2] This shows an image of cells observed after the medium was replaced with an assay buffer in Test Example 2. The gelatin concentration in the medium is shown on the left side of the photograph. [Diagram 3] 1 shows the results of measuring the activity of the olfactory receptor in Test Example 2. The vertical axis shows the fluorescence intensity. The horizontal axis shows the gelatin concentration in the medium. The legend shows the concentration of compound a. [Figure 4] This shows an image of cells observed immediately after the plate was left standing overnight at 27° C. after seeding in Test Example 3. The gelatin concentration in the medium is shown on the left side of the photograph. [Diagram 5] This shows an image of cells observed after the medium was replaced with an assay buffer in Test Example 3. The gelatin concentration in the medium is shown on the left side of the photograph. [Figure 6] 1 shows the results of measuring the activity of the olfactory receptor in Test Example 3. The vertical axis shows the fluorescence intensity. The horizontal axis shows the gelatin concentration in the medium. The legend shows the concentration of compound a. [Figure 7] This shows an image of cells observed immediately after the plate was left standing overnight at 27° C. after seeding in Test Example 4. The gelatin concentration in the medium is shown on the left side of the photograph. [Figure 8] This shows an image of cells observed after the medium was replaced with an assay buffer in Test Example 4. The gelatin concentration in the medium is shown on the left side of the photograph. [Figure 9]1 shows the results of measuring the activity of the olfactory receptor in Test Example 4. The vertical axis shows the fluorescence intensity. The horizontal axis shows the gelatin concentration in the medium. The legend shows the concentration of compound a. [Figure 10] This shows an image of cells observed immediately after the plate was left standing overnight at 27° C. after seeding in Test Example 5. The gelatin concentration in the medium is shown on the left side of the photograph. [Figure 11] This shows an image of cells observed after the medium was replaced with an assay buffer in Test Example 5. The gelatin concentration in the medium is shown on the left side of the photograph. [Figure 12] 1 shows the results of measuring the activity of the olfactory receptor in Test Example 5. The vertical axis shows the fluorescence intensity. The horizontal axis shows the gelatin concentration in the medium. The legend shows the concentration of compound a. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0021] In one aspect, the present disclosure relates to a cell substrate (sometimes referred to as the "substrate of the present disclosure" in this specification) that includes a compartment including a polypeptide-coated surface and is used to measure the activity of a sensor protein. This will be described below.

[0022] The substrate of the present disclosure is a cell substrate used for arranging (e.g., adhering) cells, and the form thereof is not particularly limited. The substrate of the present disclosure can be, for example, a cell plate, a cell chip, a cell device, a cell dish, a cell flask, a cell bag, etc.

[0023] The substrate of the present disclosure includes a compartment. The compartment is an area in which cells are arranged (e.g., adhered). The form of the compartment is not particularly limited as long as the cells can be arranged (e.g., adhered). From the viewpoint of cell desiccation resistance, cell retention, production efficiency, or chemical substance detectability, the compartment is preferably in a form divided by walls (e.g., in a well-like form and / or in a form separated from the surroundings by an outer wall). In addition, it is also possible to distinguish the surface outside the compartment from the compartment in which cells can be arranged (e.g., adhered) by making the surface on which cells cannot be arranged (e.g., adhered) (or the adhesiveness is significantly reduced).

[0024] The compartment comprises a polypeptide-coated surface (sometimes referred to herein as the "surface of the disclosure").

[0025] A polypeptide is a chain structure in which a plurality of amino acids are linked by peptide bonds, and is not particularly limited in this respect.

[0026] The amino acids constituting the polypeptide are not particularly limited, and include not only α-amino acids, but also β-amino acids, γ-amino acids, δ-amino acids, etc. Examples of α-amino acids include amino acids having a basic side chain such as lysine, arginine, and histidine, amino acids having an acidic side chain such as aspartic acid and glutamic acid, amino acids having a non-charged polar side chain such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine, amino acids having a non-polar side chain such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan, amino acids having a β-branched side chain such as threonine, valine, and isoleucine, and amino acids having an aromatic side chain such as tyrosine, phenylalanine, tryptophan, and histidine.

[0027] The number of amino acids constituting the polypeptide (the number of amino acid residues) is, for example, 3 to 5000. From the viewpoint of detectability of chemical substances, the number of amino acid residues is preferably 10 to 3000, more preferably 15 to 2000, and even more preferably 20 to 1500. In particular, in a preferred embodiment of the present disclosure, the number of amino acid residues is preferably 10 or more, more preferably 100 or more, and even more preferably 500 or more.

[0028] The polypeptides may be in the form of a single chain or a complex of two or more chains.

[0029] The polypeptide may be chemically modified as long as it has the effect of improving the detectability of chemical substances. The presence or absence of the effect of improving the detectability of chemical substances can be determined by measuring the activity of olfactory receptors using a cell substrate having a compartment including a surface coated with a test substance as described in Test Example 2 below, and comparing the results with those without coating with the test substance.

[0030] Polypeptides have a carboxyl group (-COOH) at the C-terminus, a carboxylate (-COO - ), amide (-CONH2) or ester (-COOR).

[0031] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, and n-butyl. 1-6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl groups; for example, C phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups: α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0032] The polypeptide may be amidated or esterified at a carboxyl group (or carboxylate) other than the C-terminus. In this case, the ester may be, for example, the C-terminus ester described above.

[0033] Specific examples of the polypeptide include polypeptides having cell adhesive properties or partial peptides thereof, such as extracellular matrix proteins or partial peptides thereof, and polypeptides that adhere to cells via electrostatic interactions.

[0034] Examples of extracellular matrix proteins include collagen, elastin, fibronectin, laminin, fibrinogen, fibrin, tenascin, ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs), and proteoglycan. The site and length of the partial peptide of the extracellular matrix protein are not particularly limited as long as it can adhere to cells via binding to a cell receptor. The region responsible for binding to a cell receptor is publicly known or can be easily estimated based on publicly known information. In addition, as long as it can adhere to cells via binding to a cell receptor, these extracellular matrix proteins may have a mutation (e.g., substitution, deletion, insertion, preferably conservative substitution) with respect to the wild-type amino acid sequence, and specifically, for example, may include a mutant amino acid sequence having an identity of, for example, 90% or more, 95% or more, or 99% or more with respect to the wild-type amino acid sequence.

[0035] In this specification, "conservative substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is considered to be conservative substitution. In addition, substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine are also considered to be conservative substitutions.

[0036] In this specification, the "identity" of an amino acid sequence refers to the degree of agreement between two or more comparable amino acid sequences. Thus, the higher the agreement between two amino acid sequences, the higher the identity or similarity between those sequences. The level of identity of an amino acid sequence can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87: 2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences" Proc Natl Acad Sci USA. 90: 5873-7 (1993)). Programs called BLASTP and BLASTX based on such BLAST algorithms have been developed. Specific techniques for these analysis methods are publicly known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0037] Specific examples of polypeptides that adhere to cells via electrostatic interactions include positively charged polypeptides (the ratio of positively charged amino acid residues (e.g., lysine, arginine, histidine, etc.) to the total number of amino acid residues (100%) is, for example, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100%).

[0038] As the polypeptide, from the viewpoint of enabling cells to be more stably arranged on the substrate and detect chemical substances, an extracellular matrix protein is preferable, and among them, collagen is particularly preferable.

[0039] The polypeptide may be one type alone or two or more types in combination.

[0040] The amount of the polypeptide coated on the surface of the present disclosure is not particularly limited, and may be, for example, 0.1 μg / cm 2 or more, but from the viewpoint of detectability of chemical substances, 0.5 μg / cm is preferable. 2 More preferably, 1 μg / cm 2 More preferably, 2 μg / cm 2 More preferably, 3 μg / cm 2 The upper limit of the coating amount is not particularly limited, but is, for example, 200 μg / cm 2 , 100μg / cm 2 , 50μg / cm 2 , 30μg / cm 2 , 20μg / cm 2 , 10μg / cm 2 It can be said that:

[0041] The material of the surface to be coated is not particularly limited as long as it is a material that can be coated with a polypeptide. Examples of such materials include resins (e.g., polystyrene, polyethylene, polypropylene, poly(acrylic acid ester), poly(methacrylic acid ester), polyacrylamide, polyacrylonitrile, polyethylene terephthalate, poly(L-lactic acid), poly(glycolic acid), poly(ε-caprolactone), poly(ethylene glycol), and copolymers thereof), metals (gold, silver, copper, iron, zinc, aluminum, nickel, and alloys or oxides thereof), glass, silica, silicon, and the like, or composite materials. From the viewpoint of ease of polypeptide coating, the material is preferably a resin.

[0042] The method for coating the surface to be coated with the polypeptide can be carried out according to or in accordance with a known method.

[0043] By including more of the surface of the present disclosure in the compartment, the intended effect of the present disclosure can be more effectively achieved. From this viewpoint, the area of ​​the surface of the present disclosure relative to 100% of the compartment area (area of ​​the region where cells are arranged (e.g., adhered)) is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, still more preferably 95% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0044] The area of ​​the compartment is preferably 0.5 to 100 mm from the viewpoint of detection sensitivity or production efficiency. 2 , more preferably 1 to 50 mm 2 , and more preferably 1.5 to 40 mm 2 , and even more preferably 2 to 40 mm 2 In a preferred embodiment of the present disclosure, the area is 50 mm 2 Below, 35mm 2 Less than or equal to 15mm 2 The following is the result.

[0045] From the viewpoint of detection sensitivity or production efficiency, the number of compartments contained in the substrate of the present disclosure is preferably 1 to 2000, more preferably 4 to 1600, and further preferably 8 to 400. In a preferred embodiment of the present disclosure, the number is 10 or more, 20 or more, or 50 or more.

[0046] The substrate and surface of the present disclosure can be used to measure the activity of a sensor protein of a cell. By measuring the activity of a sensor protein of a cell using the substrate and surface of the present disclosure, chemical substances can be detected with higher sensitivity. Therefore, in one aspect, the present disclosure relates to a method for measuring the activity of a sensor protein, comprising measuring the activity of the sensor protein of a cell comprising a polynucleotide comprising a coding sequence for the sensor protein arranged on the surface of the present disclosure.

[0047] When the substrate of the present disclosure is used to measure the activity of a cellular sensor protein, there is no particular limitation as long as the cells are arranged (e.g., attached) on the surface of the present disclosure. In order to prevent the cells from drying out, the compartment preferably contains a solution (e.g., a medium) or a gel (e.g., a gel containing a medium component).

[0048] From the viewpoint of detection sensitivity, a compartment usually contains multiple cells. 2 The number of cells per unit area is, for example, 50 to 20,000 cells / mm 2 From the viewpoints of detection sensitivity, cell viability, etc., the cell density is preferably 100 to 15,000 cells / mm 2 , more preferably 100 to 10,000 cells / mm 2 , and more preferably 200 to 10,000 cells / mm 2 , and even more preferably 500 to 7000 cells / mm 2 , and particularly preferably 1000 to 5000 cells / mm 2 It is.

[0049] In one embodiment of the present disclosure, the substrate of the present disclosure can be used for solution replacement or addition in a state in which cells are arranged on the surface of the present disclosure. In this case, for example, after solvent replacement or addition, it can be used to measure the activity of the sensor protein. Since a solution (such as a culture medium) or gel suitable for holding cells may contain a substance that adversely affects the detection of chemical substances or may have physical properties that may adversely affect the detection of chemical substances, it is preferable to perform solution replacement or addition before activity measurement in order to suppress the risk of such adverse effects. In the case of a gel, the gel is appropriately changed to a liquid state (for example, by increasing the temperature) before performing solution replacement or addition. This allows the solution such as a culture medium to be replaced with a solution (for example, a buffer solution) that can control the above-mentioned risk, or the above-mentioned risk can be reduced by dilution.

[0050] The substrate of the present disclosure may contain cells of only one type of sensor protein, but preferably contains cells of two or more (more preferably three or more, even more preferably four or more, even more preferably five or more, ten or more, fifteen or more, or twenty or more) types of different sensor proteins.

[0051] The cells are not particularly limited. From the viewpoint of suitability for detection of chemical substances, animal cells such as insect cells and mammalian cells are preferred, and insect cells are particularly preferred from the viewpoint of suitability for detection of chemical substances by the surface of the present invention and ease of management such as no need for CO2 or temperature management.

[0052] Examples of insect cells include Sf cells, MG1 cells, and High Five cells. TM As the Sf cells, for example, Sf9 cells (ATCC CRL1711), Sf21 cells, etc. are used. Among the insect cells, cells derived from insects of the family Arctiidae are particularly preferred.

[0053] The cells derived from an insect of the family Arctiidae are primary cultured cells or established cell lines of cells derived from an insect of the family Arctiidae, and are not particularly limited thereto.

[0054] Examples of the Arctiidae family include the Arctiinae subfamily, the Lithosiinae subfamily, and the Syntominae subfamily, and among these, the Arctiinae subfamily is preferred. Examples of the Arctiinae subfamily are preferably the genera Spilosoma, Spilarctia, and Rhagonis, and particularly preferably the Rhagonis genus. The Rhagonis genus is not particularly limited, but particularly preferably the Rhagonis genus (Spilosoma imparilis).

[0055] Cells derived from tiger moth insects can be obtained from known biological banks, or can be obtained by collecting and culturing live tiger moth insects according to or in accordance with known methods, and can also be established as lines as necessary.

[0056] Examples of cells derived from Mulberry Tree Moth include FFPRI-SpIm-2AM-SF cells (MAFF number: 275052) and FFPRI-SpIm-2AM-IPL411 cells (MAFF number: 275053) from the National Agriculture and Food Research Organization Gene Bank.

[0057] The cells preferably contain an exogenous polynucleotide that includes a coding sequence for a sensor protein, which allows the cells to express any sensor protein and increase the expression level of the target sensor protein, thereby enhancing the detection sensitivity of the target chemical substance.

[0058] An exogenous polynucleotide is a polynucleotide that contains a base sequence that is not derived from the genomic DNA (particularly chromosomal genomic DNA) of an insect cell, and is not particularly limited in this respect.

[0059] The sensor protein may be selected from proteins capable of detecting the presence of a chemical substance, and may be, for example, a receptor protein that uses a chemical substance as a ligand. The sensor protein is preferably an olfactory receptor protein, and more preferably an insect olfactory receptor protein.

[0060] Insect olfactory receptor proteins are membrane proteins with seven transmembrane structures, and act as odor sensors for living organisms. From the amino terminus (hereinafter sometimes referred to as the "N terminus") to the carboxyl terminus (hereinafter sometimes referred to as the "C terminus") of the olfactory receptor protein, the olfactory receptor protein is composed of the following linked regions in order: N-terminus region (NT), first transmembrane domain (TM1), first extracellular loop (EC1), second transmembrane domain (TM2), first intracellular loop (IC1), third transmembrane domain (TM3), second extracellular loop (EC2), fourth transmembrane domain (TM4), second intracellular loop (IC2), fifth transmembrane domain (TM5), third extracellular loop (EC3), sixth transmembrane domain (TM6), third intracellular loop (IC3), seventh transmembrane domain (TM7), and C-terminus region (CT). In the present disclosure, each region is determined by structure prediction (default conditions) using TMpred (K. Hofmann, W. Stoffel, TMbase - a database of membrane spanning proteins segments, Biol. Chem. Hoppe-Seyler, 374 (1993), p. 166, https: / / embnet.vital-it.ch / software / TMPRED_form.html).

[0061] Insects from which insect olfactory receptor proteins are derived are preferably dipteran insects such as Culicidae and Drosophilidae, lepidopteran insects such as Bombycidae, hymenopteran insects such as Apidae, orthopteran insects such as Acrididae, hemipteran insects such as Cimex, and the like, and more preferably dipteran insects such as Culicidae and Drosophilidae, orthopteran insects such as Acrididae, and hemipteran insects such as Cimex. Examples of insects from the Culicidae family include Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, and the like. Examples of insects of the Drosophilidae family include Drosophila melanogaster, Drosophila pseudoobscura, Drosophila virillis, etc. Examples of insects of the Bombyx family include Bombyx mori, Bombyx mandarina, Trilocha varians, etc. Examples of insects of the Apidae family include Apis mellifera, Apis florea, Apis dorsata, Bombus terrestris, etc. Examples of insects belonging to the Acrididae family include the migratory locust (Locusta migratoria) and the like, and examples of insects belonging to the Cimex family include the bedbug (Cimex lectularius) and the like.

[0062] Specific examples of wild-type insect olfactory receptor proteins include AaOR1, AaOR2, AaOR4, AaOR5, AaOR6, AaOR8, AaOR9, AaOR10a, AaOR15, AaOR22, AaOR24, AaOR25, AaOR26, AaOR27, AaOR28, AaOR30, AaOR34, AaOR36, AaOR38, AaOR41a, AaOR41b, AaOR42, AaOR43, AaOR44, AaOR47, AaOR49, AaOR50, AaOR52, AaOR54, AaOR58, AaOR59, and AaOR60. , AaOR61, AaOR64, AaOR65, AaOR66, AaOR67a, AaOR69a, AaOR70, AaOR71, AaOR72a, AaOR73, AaOR74, AaOR75, AaOR77, AaOR78, AaOR79, AaOR81, AaOR83b, AaOR84, AaOR85, AaOR86, AaOR87, AaOR91, AaOR95, AaOR97, AaOR96, AaOR99, AaOR100, AaOR102, AaOR103, AaOR104a, AaOR105, AaOR107, AaOR108, AaOR1 09, AaOR110, AaOR112, AaOR114, AaOR116, AaOR117, AaOR118, AaOR122, AaOR125, AaOR128, AgOR1, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR8, AgOR9, A gOR10, AgOR11a, AgOR12a, AgOR12b, AgOR13, AgOR14, AgOR15, AgOR16a, AgOR17, AgOR18, AgOR20, AgOR21, AgOR23, AgOR25, AgOR26, AgOR27, AgOR28, AgO R30, AgOR34, AgOR36, AgOR37, AgOR38, AgOR39a, AgOR40, AgOR42, AgOR44, AgOR45, AgOR46, AgOR47, AgOR49, AgOR50, AgOR54, AgOR56a, AgOR57, AgOR60 , AgOR61, AgOR62, AgOR63, AgOR64, AgOR65, AgOR69, AgOR70, AgOR71, AgOR72, AgOR74, AgOR75, AgOR76a, AmOR1, AmOR3, AmOR9, AmOR10, AmOR13, AmOR41,AmOR51, AmOR52, AmOR55, AmOR71, AmOR73, AmOR78, AmOR85, AmOR89, AmOR90, AmOR114, AmOR115, AmOR118, AmOR120, AmOR121, AmOR161, BmOR1, BmOR2, BmOR3, BmOR4, BmOR5, BmOR 8, BmOR9, BmOR10, BmOR13, BmOR17, BmOR18, BmOR23, BmOR24, BmOR25, BmOR35, BmOR36, B mOR42, BmOR45, BmOR49, BmOR51, BmOR52, BmOR55, BmOR56, BmOR61, DmOR1a, DmOR9a, DmOR 19a, DmOR22a, DmOR22b, DmOR22c, DmOR24a, DmOR30a, DmOR33a, DmOR33b, DmOR33c, DmOR 35a, DmOR42b, DmOR43a, DmOR45a, DmOR45b, DmOR47a, DmOR49b, DmOR59b, DmOR65b, DmOR6 Examples of such DmOR include DmOR5c, DmOR67b, DmOR67c, DmOR69a, DmOR71a, DmOR74a, DmOR82a, DmOR83a, DmOR83c, DmOR85a, DmOR85c, DmOR85e, DmOR85f, DmOR88a, DmOR92a, DmOR94a, DmOR94b, and DmOR98b.

[0063] In the present specification, OR indicates an olfactory receptor, Dm indicates that it is derived from Drosophila melanogaster, Bm indicates that it is derived from Bombyx mori, Ag indicates that it is derived from Anopheles gambiae, and Aa indicates that it is derived from Aedes aegypti. The amino acid sequences and coding sequences of various olfactory receptor proteins including these are publicly known or can be easily identified by a sequence identity search based on the publicly known sequences.

[0064] The sensor protein may contain amino acid mutations relative to the wild-type amino acid sequence, so long as the chemical response activity is not significantly reduced. "Not significantly reduced" means, for example, that the chemical response activity of the sensor protein containing the amino acid mutation is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and even more preferably 90% or more, relative to 100% of the chemical response activity of the wild-type sensor protein.

[0065] The amino acid mutation is, for example, an amino acid substitution, insertion, addition or deletion, preferably a substitution, particularly preferably a conservative substitution.

[0066] The sensor protein can include a wild-type amino acid sequence, or an amino acid sequence that has, for example, 70% or more identity to the wild-type amino acid sequence, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more identity.

[0067] In this specification, the chemical response activity refers to the property that a sensor protein recognizes a chemical substance and exhibits a signal transduction activity (e.g., an ion channel activity) either alone or in conjunction with another protein. When the sensor protein is an olfactory receptor, It may be a G protein-coupled receptor or an ion channel type receptor, but in the case of an insect olfactory receptor, it refers to a property in which an olfactory receptor recognizes a chemical substance, and an olfactory receptor complex formed by the olfactory receptor and the olfactory receptor co-receptor is activated to exhibit ion channel activity. The chemical substance response activity of a sensor protein can be measured using as an index (for example, by quantifying and evaluating the amount of signal molecules) the signal transduction activity of a sensor protein that has come into contact with a chemical substance. In the case of an insect olfactory receptor, the chemical substance response activity of an olfactory receptor can be measured using as an index the ion channel activity of an olfactory receptor complex formed by an olfactory receptor and an olfactory receptor co-receptor that have come into contact with a chemical substance. For example, a chemical substance is contacted with a cell expressing a protein that emits fluorescence or luminescence due to ions (such as calcium ions) that flow into the cell when (a) an olfactory receptor, (b) an olfactory receptor co-receptor, and (c) an olfactory receptor complex respond, and the amount of luminescence of the cell is measured. The greater the amount of luminescence measured, the higher the chemical substance response activity of the olfactory receptor is determined to be. Specifically, the measurement can be performed according to the method described in Patent Document 1.

[0068] The coding sequence of the sensor protein is not particularly limited as long as it is a base sequence that codes for the sensor protein. In one embodiment, the exogenous polynucleotide includes an expression cassette for the sensor protein. The expression cassette is not particularly limited as long as it is a polynucleotide that can express the sensor protein in a cell. A typical example of an expression cassette for a sensor protein is a polynucleotide that includes a promoter and a coding sequence for the sensor protein placed under the control of the promoter.

[0069] When the sensor protein is an insect olfactory receptor, the exogenous polynucleotide preferably contains a coding sequence for an insect olfactory receptor co-receptor. The insect olfactory receptor co-receptor is a membrane protein having a seven-transmembrane structure similar to the olfactory receptor, and functions by forming a heterocomplex with the olfactory receptor. The olfactory receptor complex, which is a heterocomplex composed of an olfactory receptor and an olfactory receptor co-receptor, has ion channel activity that is activated by odorants, and when activated, it transports sodium ions (Na + ), calcium ion (Ca 2+ ) into the cell.

[0070] The exogenous polynucleotide preferably contains a coding sequence for a protein that emits fluorescence or luminescence due to ions (such as calcium ions) that flow into the cell when a sensor protein (particularly an olfactory receptor protein) responds. Examples of such proteins include aequorin, Yellow Cameleon (YC), GCaMP, etc. Alternatively, the cell of the present disclosure preferably contains an ion-dependent fluorescent dye such as a calcium ion-dependent fluorescent dye (e.g., Fura-2, Fluo-3, Fluo-4, etc.).

[0071] Coding sequences such as the coding sequence of an insect olfactory receptor co-receptor, the coding sequence of a fluorescent or luminescent protein, and the coding sequence of a drug resistance gene are preferably contained in the foreign polynucleotide in the form of an expression cassette. The structure of the expression cassette is the same as that of the expression cassette of the sensor protein. The promoter of the expression cassette can be shared among multiple coding sequences.

[0072] The exogenous polynucleotide is preferably integrated into genomic DNA (particularly preferably chromosomal genomic DNA). This allows stable expression of the sensor protein, making it suitable for chemical detection. In this case, the polynucleotide can be one continuous region in the genomic DNA, or a combination of two or more continuous regions (for example, a form in which the sensor protein coding sequence is included in continuous region A and a drug resistance gene coding sequence is included in continuous region B, which is a continuous region other than continuous region A, or a form in which the sensor protein coding sequence is included in both continuous region A and continuous region B).

[0073] In another embodiment, the polynucleotide may be in a state where it is not integrated into genomic DNA. In this case, the exogenous polynucleotide may be in the form of, for example, a vector. In this case, the polynucleotide may be one type of polynucleotide molecule, or may be two or more types of polynucleotide molecules (for example, a form in which a sensor protein coding sequence is contained in polynucleotide molecule A and a drug resistance gene coding sequence is contained in polynucleotide molecule B, which is a molecule other than polynucleotide molecule A, or a form in which a sensor protein coding sequence is contained in both polynucleotide molecule A and polynucleotide molecule B).

[0074] By measuring the activity of the sensor protein, a chemical substance can be detected. The chemical substance can be a chemical substance in a sample such as a body fluid (e.g., urine, blood, saliva), air (e.g., indoor air, air in a package), or water (e.g., river water, seawater, tap water, clean water, sewage). In this case, for example, by adding a sample to a compartment of the substrate of the present disclosure, the chemical substance in the sample can reach the cell and come into contact with the sensor protein of the cell. For example, by detecting ions flowing into the cell (for example, by detecting using a protein that emits fluorescence or luminescence in response to ions), the activity of the sensor protein can be measured and the chemical substance can be detected. EXAMPLES

[0075] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0076] Test Example 1. Preparation of stable expression cells A transposon vector in which an olfactory receptor protein (ORA) coding sequence, an olfactory receptor co-receptor coding sequence, a calcium sensor fluorescent protein coding sequence, and a puromycin resistance gene coding sequence are arranged under the control of a promoter sequence and between the 5'ITR (inverted repeat) and 3'ITR was introduced into a cell derived from the moth (Spilosoma imparilis) and selected with puromycin to produce an olfactory receptor stably expressing SpIm cell (hereinafter referred to as ORA cell) in which foreign DNA containing the coding sequence and promoter sequence is integrated into chromosomal genomic DNA. The olfactory receptor is an insect-derived olfactory receptor and is a receptor for compound a. ORA cells emit fluorescence in response to compound a.

[0077] Test Example 2. Measurement of olfactory receptor activity (TC: Tissue Culture) ORA cells were detached from the culture flask and mixed with medium (Sf-900 III, Gibco) containing 0–2% fish gelatin to prepare a cell suspension. The cell density of the cell suspension was counted using a cell counter. When the cell density was 5 × 10 5 ~1×10 6 The cell density was adjusted to 100 cells / mL. Then, using a dispenser (ASSIST PLUS, Integra), cells were seeded into a TC-treated 384-well plate (Greiner, catalog number: 781098) so that the same number of cells per well (40 μL / well) was obtained. The cell seeding density is shown in Table 1. After seeding, the plate was left to stand overnight at 27°C to allow the cells to adhere to the plate. The state of cell adhesion was observed under a microscope. The observed image is shown in Figure 1. The plate was then stored at 4°C.

[0078] The plate was stored at 4°C for 3 days and then left to stand at 27°C for 2 hours. The medium was then replaced with assay buffer (Hanks' buffer containing 0.1% BSA and 20 mM HEPES) using a dispenser (40 μL / well). The cells were then observed under a microscope. The observed images are shown in Figure 2.

[0079] Compound a (a substance to which ORA responds) solution or a solution identical to compound a solution except that it did not contain compound a was added to each well so that the final concentrations were 0, 0.1, 1, and 10 μM, and the change in fluorescence intensity before and after the addition was measured using a microplate reader (FlexStation3, Molecular Devices). The results are shown in Figure 3.

[0080] When the solution is replaced while the cells are attached, mechanical stress is applied to the cells. This causes the cells to detach, especially in wells containing gelatin (Figure 2). Although cell detachment was not evident in wells that did not contain gelatin (Figure 2), it is thought that this may occur with a certain frequency. Furthermore, the detectability of chemical substances decreased in wells that contained gelatin (0.1 μM compound a could not be detected (Figure 3)).

[0081] Test Example 3. Measurement of olfactory receptor activity (Amine coat) From the results of Test Example 2, it was considered that the decrease in the detectability of chemical substances in wells containing gelatin could be suppressed by improving cell adhesion. Therefore, a test was performed in the same manner as Test Example 2 using a 384-well plate coated with a coating (Amine Coat, Falcon, catalog number: 356719) that improves cell adhesion. After seeding the cells, the plate was left to stand overnight at 27°C. The image of the cells observed immediately after the plate was left to stand overnight is shown in FIG. 4, the image of the cells observed after the medium was replaced with an assay buffer is shown in FIG. 5, and the results of the measurement of olfactory receptor activity are shown in FIG. 6.

[0082] Although amine coating improved cell adhesion (compare Figures 2 and 4), 0.1 μM compound a could not be detected (Figure 6), and chemical detection was not improved.

[0083] Test Example 4. Measurement of olfactory receptor activity (Poly-D-Lysine coating) Given the results of Test Example 3, we decided to try a different coating. Using a Poly-D-Lysine-coated 384-well plate (Falcon, catalog number: 356660), we conducted a test in the same manner as Test Example 2. Figure 7 shows an image of the cells immediately after the plate was left to stand overnight at 27°C after seeding the cells, Figure 8 shows an image of the cells after replacing the medium with assay buffer, and Figure 9 shows the results of measuring the activity of the olfactory receptors.

[0084] The poly-D-lysine coating showed the same improvement in cell adhesion as the amine coating (compare Figures 4 and 8). However, unexpectedly, compound a was detected at 0.1 μM (Figure 9), confirming the improvement in chemical detection.

[0085] Test Example 5. Measurement of olfactory receptor activity (collagen coating) Based on the results of Test Example 4, it was thought that the detectability of chemical substances could be improved by coating with a polypeptide. Therefore, a test similar to Test Example 2 was carried out using a 384-well plate (Falcon, catalog number: 356702) coated with collagen, which is a polypeptide. Figure 10 shows an image of the cells immediately after the plate was left to stand overnight at 27°C after seeding the cells, Figure 11 shows an image of the cells after replacing the medium with an assay buffer, and Figure 12 shows the results of measuring the activity of the olfactory receptors.

[0086] Similar to the Poly-D-Lysine coating, the collagen coating was able to detect 0.1 μM of compound a (Figure 12), confirming the improvement in chemical detection. In particular, the collagen coating caused less cell detachment (Figure 11), suggesting that detection was more stable.

Claims

1. A cell substrate for use in measuring the activity of a sensor protein, comprising a compartment comprising a polypeptide-coated surface.

2. The cell substrate according to claim 1 , wherein the polypeptide is an extracellular matrix protein or a partial peptide thereof.

3. The cell substrate according to claim 1 , wherein the polypeptide is collagen.

4. The coating amount of the polypeptide is 0.5 μg / cm 2 The cell substrate according to claim 1 , wherein the cell substrate is a substrate for cells.

5. The area of ​​the compartment is 1 to 50 mm 2 The cell substrate according to claim 1 ,

6. The cell substrate according to any one of claims 1 to 5, wherein the cells contain a polynucleotide comprising a coding sequence for a sensor protein.

7. The cell substrate according to claim 6 , wherein the sensor protein is an olfactory receptor protein.

8. 6. The cell substrate according to claim 1, which is used to measure the activity of the sensor protein after replacing or adding a solution with cells disposed on the surface.

9. The cell substrate according to any one of claims 1 to 5, wherein the cells are insect cells.

10. A method for measuring the activity of a sensor protein comprising measuring the activity of said sensor protein in cells disposed on a polypeptide-coated surface.

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