Insect-derived cells of sphagnaceae family

Abstract

Provided is a cell which can be used in a chemical substance sensor and has excellent cold storage resistance. A cell of insect origin of the Cordyceps family comprising an exogenous polynucleotide comprising a coding sequence of a sensor protein.

Description

TECHNICAL FIELD

[0001] The present application relates to cells and the like derived from insects of the family Erebidae. BACKGROUND

[0002] A group of odor substances that characterize a specific disease, mental state, and the like of a human being has been identified, and various odor sensors that target them have become popular because of their high value as diagnostic markers. Since biological olfactory receptors have superior characteristics such as diversity, sensitivity, selectivity, and the like that semiconductor and other odor sensor elements of the past do not have, development of new odor sensors that use olfactory receptors as sensor elements is expected.

[0003] In Patent Literature 1, a cell that expresses an altered olfactory receptor, and a lipid bilayer membrane that has an altered olfactory receptor are disclosed as odor sensors.

[0004] PRIOR ART DOCUMENTS

[0005] PATENT LITERATURE

[0006] Patent Literature 1: International Publication No. 2022 / 024902 SUMMARY

[0007] PROBLEMS TO BE SOLVED BY THE INVENTION

[0008] An odor sensor that includes a step of artificially preparing a lipid bilayer membrane that has a sensor protein such as an olfactory receptor is not necessarily sufficient in terms of production efficiency, and further improvement in production efficiency of odor sensors is required. Therefore, attention is focused on the use of a cell that expresses a sensor protein.

[0009] In the case of using a cell as a chemical substance sensor such as an odor sensor, from the viewpoint of ease of use, a form in which the cell is not used by culturing and preparing it at all times, but is prepared in advance and kept in a container or the like, and is used when needed, is desired. In the case of the latter form, the storage resistance of the cell is required, and from the viewpoint of not drying the cell, cold resistance is particularly important.

[0010] The present disclosure aims to provide a cell that has excellent cold resistance and can be used in a chemical substance sensor.

[0011] MEANS FOR SOLVING THE PROBLEMS

[0012] The present inventors conducted intensive studies in view of the above problems, and as a result, found that a cell derived from an insect of the family Erebidae has excellent cold resistance and can be used in a chemical substance sensor. The present inventors conducted further studies based on this knowledge, and as a result, completed the present disclosure. That is, the present disclosure includes the following aspects.

[0013] Item 1. A cell derived from an insect of the family Arctiidae, which comprises an exogenous polynucleotide comprising a coding sequence of a sensor protein.

[0014] Item 2. The cell according to Item 1, wherein the aforementioned sensor protein is an olfactory receptor protein.

[0015] Item 3. The cell according to Item 1 or 2, wherein the aforementioned sensor protein is an insect olfactory receptor protein.

[0016] Item 4. The cell according to any one of Items 1 to 3, wherein the aforementioned exogenous polynucleotide comprises a coding sequence of an olfactory receptor co-receptor protein, and a coding sequence of a chromogenic or luminescent protein.

[0017] Item 5. The cell according to any one of Items 1 to 4, wherein the aforementioned exogenous polynucleotide comprises a coding sequence of a drug resistance gene.

[0018] Item 6. The cell according to any one of Items 1 to 5, wherein the aforementioned exogenous polynucleotide is integrated into genomic DNA.

[0019] Item 7. The cell according to any one of Items 1 to 6, wherein the insect of the family Arctiidae is an insect of the genus Arctia.

[0020] Item 8. The cell according to Item 7, wherein the insect of the genus Arctia is Arctia caja.

[0021] Item 9. A cell having cold storage resistance, which comprises an exogenous polynucleotide comprising a coding sequence of a sensor protein.

[0022] Item 10. The cell according to any one of Items 1 to 9, which is determined to have cold storage resistance in a case where the following cold storage resistance test is performed.

[0023] < Cold storage resistance test >

[0024] Step (1): The cell is inoculated at 1 x 10 5 / 200 μL / well in a plate having a partition, and left to stand at 27°C for 6 hours,

[0025] Step (2): The plate is covered with aluminum foil to shield from light, and left to stand at 4°C for 72 hours, and then the fluorescence intensity is measured.

[0026] Step (3): After a substance to which the sensor protein responds is added to each well, the fluorescence intensity is measured.

[0027] Step (4): In a case where the fluorescence intensity measured in Step (3) is greater than the fluorescence intensity measured in Step (2), the cell is determined to have cold storage resistance.

[0028] Item 11. The cell according to any one of Items 1 to 10, which has a survival rate of 50% or more after being stored at 4°C for 15 days.

[0029] Item 12. A cell chip comprising a partition containing the cells according to any one of Items 1 to 11.

[0030] Item 13. The cell chip according to Item 12, which is used for detecting chemical substances.

[0031] Effects of the Invention

[0032] According to the present disclosure, it is possible to provide cells having excellent cold storage resistance that can be used in chemical substance sensors, and a cell chip using the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] [ Figure 1 ] shows the cell viability measured in Test Example 1. The vertical axis shows the cell viability (Day 0 is 100%), and the horizontal axis shows the storage time at 4°C. The legend shows the cells used.

[0034] [ Figure 2 ] shows the chemical response activity of ORA cells measured in Experiment 2. The vertical axis shows the difference in fluorescence intensity before and after addition of the chemical (Compound a), and the horizontal axis shows the concentration of the chemical. The left graph shows the results for the case of non-refrigerated storage, and the right graph shows the results for the case of refrigerated storage.

[0035] [ Figure 3 This figure shows the chemical response activity of SpIm cells stably expressing olfactory receptors measured in Experimental Example 3. The top of the graph shows the olfactory receptors expressed by the cells, and the bottom of the graph shows the chemical substances used. The vertical axis shows the difference in fluorescence intensity before and after addition of the chemical substance, and the horizontal axis shows the concentration of the chemical substance.

[0036] [ Figure 4 This figure shows the chemical response activity of SpIm cells stably expressing olfactory receptors measured in Experimental Example 3. The top of the graph shows the olfactory receptors expressed by the cells, and the bottom of the graph shows the chemical substances used. The vertical axis shows the difference in fluorescence intensity before and after addition of the chemical substance, and the horizontal axis shows the concentration of the chemical substance. DETAILED DESCRIPTION

[0037] In this specification, the expressions “comprise” and “include” include the concepts of “comprise”, “include”, “consist essentially of” and “consist only of”.

[0038] In one embodiment, the present disclosure relates to cells derived from insects of the family Tetrapodidae (sometimes referred to herein as "cells of the present disclosure"), comprising an exogenous polynucleotide containing a coding sequence for a sensor protein. This will be described below.

[0039] The insect-derived cell of the Arctiidae is a primary culture cell or a strain cell of an organism constituting cell of the insect-derived cell of the Arctiidae, and is not particularly limited in this definition.

[0040] As the Arctiidae, for example, Arctiinae, Lithosiinae, Syntominae, and the like are exemplified, and among them, Arctiinae is preferably exemplified. As the Arctiinae, the genera of Spilosoma, Spilarctia, and Rhagonis are preferably exemplified, and Spilosoma is particularly preferably exemplified. As the Spilosoma, there is no particular limitation, and from the viewpoint of cold storage tolerance and the like, Spilosoma imparilis is particularly preferably exemplified.

[0041] The insect-derived cell of the Arctiidae can be obtained from a known biological bank, and can be collected / cultured from the organism of the insect of the Arctiidae according to or based on a known method, and can be obtained as a strain as needed.

[0042] As the Spilosoma imparilis-derived cell, for example, FFPRI-SpIm-2AM-SF cell (MAFF No. 275052), FFPRI-SpIm-2AM-IPL411 cell (MAFF No. 275053), and the like of the Agricultural Biological Resource Center are exemplified.

[0043] The cell of the present disclosure is an insect-derived cell of the Arctiidae, and is a cell containing an exogenous polynucleotide containing a coding sequence of a sensor protein.

[0044] The exogenous polynucleotide is a polynucleotide containing a base sequence not from the genomic DNA (particularly, chromosomal genomic DNA) of the insect-derived cell of the Arctiidae, and is not particularly limited in this definition.

[0045] In the present specification, polynucleotides are exemplified by, in addition to typical polynucleotides such as DNA and RNA in vivo, polynucleotides subjected to known chemical modifications, artificial polynucleotides, and the like. In order to prevent decomposition by hydrolytic enzymes such as nucleases, the phosphate residue (phosphoric acid ester) of each nucleotide can be replaced with, for example, a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, phosphorodithioate, and the like. Furthermore, the hydroxyl group at position 2 of the sugar (ribose) of each ribonucleotide can be replaced with -OR (R represents, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, and the like). Further, the nucleic acid base moiety (pyrimidine, purine) can be chemically modified, for example, by the introduction of a methyl group, a cationic functional group, or the like to position 5 of the pyrimidine base, or the substitution of the carbonyl group at position 2 with a thiocarbonyl group, and the like. Further, the phosphate moiety, the hydroxyl moiety, and the like can be modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, and the like, but are not limited thereto. Furthermore, BNA (LNA) and the like, in which the conformation of the sugar moiety is fixed to the N type by cross-linking the 2' oxygen and the 4' carbon of the sugar moiety of the nucleotide, can also be applied.

[0046] The sensor protein can be selected from among proteins that can detect the presence of a chemical substance, and can be, for example, a receptor protein that uses a chemical substance as a ligand. The sensor protein is particularly preferably an olfactory receptor protein.

[0047] The olfactory receptor protein is a membrane protein having a 7-transmembrane structure, and functions as a biological odor sensor. In order from the amino-terminal end (hereinafter, sometimes also referred to as "N-terminal end") to the carboxyl-terminal end (hereinafter, sometimes also referred to as "C-terminal end") of the olfactory receptor protein, an N-terminal region (NT), a 1st transmembrane domain (TM1), a 1st extracellular loop (EC1), a 2nd transmembrane domain (TM2), a 1st intracellular loop (IC1), a 3rd transmembrane domain (TM3), a 2nd extracellular loop (EC2), a 4th transmembrane domain (TM4), a 2nd intracellular loop (IC2), a 5th transmembrane domain (TM5), a 3rd extracellular loop (EC3), a 6th transmembrane domain (TM6), a 3rd intracellular loop (IC3), a 7th transmembrane domain (TM7), and a C-terminal region (CT) are linked to constitute. In the present disclosure, each region is determined by applying the structure prediction of TMpred (K. Hofmann, W. Stoffel, TMbase - a database of membrane-spanning protein segments, Biol. Chem. Hoppe-Seyler, 374 (1993), p. 166, https: / / embnet.vital-it.ch / software / TMPRED_form.html) (under the condition of default).

[0048] From the viewpoint of suitability for detection of a chemical substance, insect olfactory receptor proteins are particularly preferable. As the source insect of the insect olfactory receptor proteins, the following are preferably listed: dipteran insects such as mosquitoes and fruit flies; lepidopteran insects such as silkworm moths; hymenopteran insects such as bees; orthopteran insects such as locusts; hemipteran insects such as bed bugs; and the like, and further, dipteran insects such as mosquitoes and fruit flies; orthopteran insects such as locusts; hemipteran insects such as bed bugs are further preferably listed. As the insect of the mosquito family, for example, Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, and the like are listed. As the insect of the fruit fly family, for example, Drosophila melanogaster, Drosophila pseudoobscura, Drosophila virillis, and the like are listed. As the insect of the silkworm moth family, for example, Bombyx mori, Bombyx mandarina, Trilocha varians, and the like are listed. As the insect of the bee family, for example, Apis mellifera, Apis florea, Apis dorsata, Bombus terrestris, and the like are listed. As the insect of the locust family, for example, Locusta migratoria and the like are listed, and as the insect of the bed bug family, for example, Cimex lectularius and the like are listed.

[0049] As insect olfactory receptor proteins of wild type, specifically, for example, AaOR1, AaOR2, AaOR4, AaOR5, AaOR6, AaOR7, 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, 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, AaOR109, AaOR110, AaOR112, AaOR114, AaOR116, AaOR117, AaOR118, AaOR122, AaOR125, AaOR128, AgOR1, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR7, AgOR8, AgOR9, AgOR10, AgOR11a, AgOR12a, AgOR12b, AgOR13, AgOR14, AgOR15, AgOR16a, AgOR17, AgOR18, AgOR20, AgOR21, AgOR23, AgOR25, AgOR26, AgOR27, AgOR28, AgOR30, 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, BmOR8, BmOR9, BmOR10, BmOR13, BmOR17, BmOR18, BmOR23, BmOR24, BmOR25, BmOR35, BmOR36, BmOR42, BmOR45, BmOR49, BmOR51, BmOR52, BmOR55, BmOR56, BmOR61, DmOR1a, DmOR9a, DmOR19a, DmOR22a, DmOR22b, DmOR22c, DmOR24a, DmOR30a, DmOR33a, DmOR33b, DmOR33c, DmOR35a, DmOR42b, DmOR43a, DmOR45a, DmOR45b, DmOR47a, DmOR49b, DmOR59b, DmOR65b, DmOR65c, DmOR67b, DmOR67c, DmOR69a, DmOR71a, DmOR74a, DmOR82a, DmOR83a, DmOR83b, DmOR83c, DmOR85a, DmOR85c, DmOR85e, DmOR85f, DmOR88a, DmOR92a, DmOR94a, DmOR94b, DmOR98b, and the like.

[0050] In the present specification, OR indicates an odorant receptor, Dm indicates a Drosophila melanogaster origin, Bm indicates a Bombyx mori origin, Ag indicates an Anopheles gambiae origin, and Aa indicates an Aedes aegypti origin. The amino acid sequences and coding sequences of various odorant receptor proteins including these are known or can be easily identified by a sequence homology search based on the known sequences.

[0051] The sensor protein can include an amino acid mutation to the wild-type amino acid sequence as long as the chemical substance response activity is not significantly reduced. "Not significantly reduced" means, for example, that the chemical substance response activity of the sensor protein including the amino acid mutation is 50% or more, preferably 60% or more, more preferably 70% or more, further preferably 80% or more, and still further preferably 90% or more, relative to the chemical substance response activity of the wild-type sensor protein.

[0052] The amino acid mutation is, for example, substitution, insertion, addition, or deletion of an amino acid, preferably substitution, and particularly preferably conservative substitution.

[0053] In the present specification, "conservative substitution" means substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution of an amino acid residue having a basic side chain such as lysine, arginine, histidine, and the like with each other corresponds to a conservative substitution. In addition, substitution of an amino acid residue having an acidic side chain such as aspartic acid, glutamic acid, and the like; an amino acid residue having a non-charged polar side chain such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and the like; an amino acid residue having a non-polar side chain such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and the like; an amino acid residue having a β-branched side chain such as threonine, valine, isoleucine, and the like; an amino acid residue having an aromatic side chain such as tyrosine, phenylalanine, tryptophan, histidine, and the like with each other also corresponds to a conservative substitution.

[0054] The sensor protein can comprise a wild-type amino acid sequence, an amino acid sequence having, for example, 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, more further preferably 98% or more, particularly preferably 99% or more, of identity with respect to the wild-type amino acid sequence.

[0055] In the present specification, "identity" of an amino acid sequence means the degree of identity of two or more comparable amino acid sequences with respect to each other. Thus, the higher the identity of two amino acid sequences, the higher the identity or similarity of their sequences. The level of identity of an amino acid sequence is determined, for example, using FASTA, a tool for sequence analysis, with default parameters. Alternatively, it can be determined using the algorithm BLAST of 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)). A program called BLASTX based on such BLAST-based algorithm has been developed. The specific methods of these analysis methods are well known and can be referred to, for example, the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ).

[0056] The sensor protein can be added with other amino acid sequences, such as protein tags, fluorescent proteins, luminescent proteins, signal sequences, and the like, as long as the chemical substance-responsive activity is not significantly impaired. As a protein tag, for example, biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, and the like are exemplified.

[0057] In the present specification, the chemical substance-responsive activity of a sensor protein refers to the property of the sensor protein recognizing a chemical substance, the sensor protein alone or in combination with other proteins exhibiting signal transduction activity (e.g., ion channel activity). In the case of an olfactory receptor, it refers to the property of the olfactory receptor recognizing a chemical substance, the olfactory receptor activating an olfactory receptor complex formed with an olfactory receptor co-receptor to exhibit ion channel activity. The chemical substance-responsive activity of a sensor protein can be determined by taking the signal transduction activity of the sensor protein in contact with a chemical substance as an index (e.g., quantifying / evaluating the amount of a signal molecule). In the case of an olfactory receptor, the chemical substance-responsive activity of the olfactory receptor can be determined by taking the ion channel activity of an olfactory receptor complex formed with an olfactory receptor co-receptor in contact with a chemical substance as an index. For example, in the case of (a) an olfactory receptor, (b) an olfactory receptor co-receptor, and (c) an olfactory receptor complex, a cell expressing a protein that exhibits color development or luminescence by the influx of ions (calcium ions, etc.) into the cell is brought into contact with a chemical substance, and the amount of luminescence of the cell is determined. The more the amount of luminescence determined, the higher the chemical substance-responsive activity of the olfactory receptor is determined to be. Specifically, it can be determined according to the method described in Patent Literature 1.

[0058] The coding sequence of a sensor protein is not particularly limited as long as it is a base sequence encoding a sensor protein. The exogenous polynucleotide comprises, in one embodiment thereof, an expression cassette of a sensor protein. The expression cassette is not particularly limited as long as it is a polynucleotide that can express a sensor protein in a cell. As a typical example of an expression cassette of a sensor protein, a polynucleotide comprising a promoter and a coding sequence of a sensor protein disposed under the control of the promoter is exemplified.

[0059] The promoter is not particularly limited and can be appropriately selected. As the promoter, for example, various pol II-based promoters can be used. The pol II-based promoter is not particularly limited, and for example, a CMV promoter, an EF1 promoter, an SV40 promoter, an MSCV promoter, a promoter of a gene of insect origin, and the like are exemplified.

[0060] In the case where the sensor protein is an insect olfactory receptor, from the viewpoint that the cell of the present disclosure can be used as it is in chemical substance detection use, the exogenous polynucleotide preferably comprises a coding sequence of an olfactory receptor co-receptor of an insect. The olfactory receptor co-receptor of an insect is a membrane protein having a 7-transmembrane structure like an olfactory receptor, but it does not recognize odorants by itself and functions as a hetero complex with an olfactory receptor. The olfactory receptor complex formed with an olfactory receptor and an olfactory receptor co-receptor as a hetero complex has ion channel activity activated by odorants, and upon activation, cations such as sodium ions (Na + ), calcium ions (Ca 2+ ), etc. flow into the cell.

[0061] From the viewpoint that the cells of the present disclosure can be used as they are in chemical substance detection use, the exogenous polynucleotide preferably contains a coding sequence of a protein that develops color or emits light in response to a sensor protein (particularly, an olfactory receptor protein) by ions (calcium ions, etc.) flowing into the cell. As such a protein, Aequorin, Yellow Cameleon (YC), GCaMP, etc. are listed. Alternatively, the cells of the present disclosure preferably contain a calcium ion-dependent fluorescent dye (e.g., Fura-2, Fluo-3, Fluo-4, etc.) or the like ion-dependent fluorescent dye.

[0062] From the viewpoint that the cells of the present disclosure can be used as they are in chemical substance detection use, the cells of the present disclosure contain a sensor protein, i.e., a sensor protein is expressed in the cells of the present disclosure. In one mode, the sensor protein has a 7-transmembrane structure, and thus is disposed on the cell membrane as a membrane protein.

[0063] In order that the cells of the present disclosure can be used for drug screening, the exogenous polynucleotide preferably contains a coding sequence of a drug resistance gene. As the drug resistance gene, a gene resistant to a drug that can be used in drug screening of insect cells is selected, and for example, chloramphenicol resistance gene, tetracycline resistance gene, neomycin resistance gene, erythromycin resistance gene, phleomycin resistance gene, kanamycin resistance gene, hygromycin resistance gene, puromycin resistance gene, etc. are listed.

[0064] The coding sequence of the olfactory receptor co-receptor of the insect, the coding sequence of the protein that develops color or emits light, the coding sequence of the drug resistance gene, etc. are preferably contained in the exogenous polynucleotide in the form of an expression cassette. As for the constitution of the expression cassette, the same as the expression cassette of the sensor protein. The promoter of the expression cassette can be shared among the plurality of coding sequences.

[0065] The exogenous polynucleotide is preferably integrated into the genomic DNA (particularly preferably, chromosomal genomic DNA). Thereby, the sensor protein can be stably expressed, and suitable for chemical substance detection. In this case, the exogenous polynucleotide can be one continuous region within the genomic DNA, and in addition, can be a combination of two or more continuous regions (for example, the sensor protein coding sequence is contained in a continuous region A, the coding sequence of the drug resistance gene is contained in a continuous region B that is a continuous region different from the continuous region A, or the sensor protein coding sequence is contained in both the continuous region A and the continuous region B).

[0066] The exogenous polynucleotide can be, in another aspect, in a state of not being integrated into the genomic DNA. In this case, the exogenous polynucleotide can be, for example, in the form of a vector. In this case, the exogenous polynucleotide can be one polynucleotide molecule, and can also be two or more polynucleotide molecules (for example, the sensor protein-encoding sequence is contained in polynucleotide molecule A, the coding sequence of a drug resistance gene is contained in polynucleotide molecule B which is a different molecule from polynucleotide molecule A, or the sensor protein-encoding sequence is contained in both polynucleotide molecule A and polynucleotide molecule B).

[0067] The present disclosure, in one aspect thereof, relates to a cell having cold storage tolerance (the cell is sometimes also referred to as "a cell of the present disclosure"), which comprises an exogenous polynucleotide containing a coding sequence of a sensor protein.

[0068] In one aspect of the present application, the cold storage tolerance can be determined based on the following Cold Storage Tolerance Test 1:

[0069] < Cold Storage Tolerance Test 1 >

[0070] Step (1): Cells are inoculated at 1 x 10 5 / 200 μL / zone in a plate having zones, and left to stand at 27°C for 6 hours,

[0071] Step (2): The plate is shielded from light with aluminum foil, and after left to stand at 4°C for 72 hours, the fluorescence intensity is measured,

[0072] Step (3): After adding a substance to which the sensor protein responds to each zone, the fluorescence intensity is measured,

[0073] Step (4): In the case where the fluorescence intensity measured in Step (3) is greater than the fluorescence intensity measured in Step (2), the cell is determined to have cold storage tolerance.

[0074] More detailed conditions regarding the Cold Storage Tolerance Test 1 are described in Test Example 2 described later.

[0075] In one aspect of the present application, the cold storage tolerance can be defined as a survival rate of a certain level or more after storage at 4°C for a certain period of time. For example, the cold storage tolerance can be a survival rate of, for example, 50% or more, preferably 60% or more, more preferably 70% or more, and further preferably 80% or more after storage at 4°C for 14 days. The cold storage tolerance is measured according to Test Example 1 described later.

[0076] From the viewpoint of being suitable for a chemical substance sensor, the cell of the present disclosure is preferably in the form of being contained in a cell chip. Thus, the present disclosure, in one aspect thereof, relates to a cell chip (in the present specification, sometimes also referred to as "a cell chip of the present disclosure"), which comprises a zone containing a cell of the present disclosure.

[0077] The partition of the cell chip of the present disclosure preferably contains cells and a hydrogel. Thereby, drying of the cells can be prevented.

[0078] The form of the partition is not particularly limited as long as it is a form that can hold cells. From the viewpoints of the holding property of the cells, the efficiency of preparation, or the sensitivity of chemical substance detection, the partition is preferably a well.

[0079] The material of the partition is not particularly limited as long as it can hold cells. The material can be, for example, resin, metal, or the like.

[0080] From the viewpoint of the sensitivity of detection, the partition generally contains a plurality of cells. The number of cells per area (cm 2 ) of the partition is, for example, 1 x 10 3 to 1 x 10 9 cells / cm 2 .

[0081] From the viewpoint of the sensitivity of detection, or the viewpoint of the efficiency of preparation, the bottom area of 1 partition is preferably 0.5 to 100 mm 2 , more preferably 1 to 30 mm 2 , further preferably 1.5 to 10 mm 2 .

[0082] From the viewpoint of the sensitivity of detection, or the viewpoint of the efficiency of preparation, the number of partitions contained in the cell chip is preferably 10 to 2000, more preferably 30 to 1000, further preferably 50 to 500.

[0083] The cells of the present disclosure are excellent in cold storage resistance. In the present specification, "cold storage resistance" means that the cell survival rate, and / or the chemical substance-responsive activity is maintained in a higher state after a certain period of time (for example, 1 day or more, 1 to 30 days, 2 to 20 days, 3 to 10 days) under cold storage (for example, 0 to 10°C, 0 to 8°C, 0 to 6°C, 2 to 6°C). The cell survival rate, the chemical substance-responsive activity can be measured according to or based on the method of the Examples described later.

[0084] The cells of the present disclosure are excellent in the chemical substance-responsive activity using a sensor protein compared to cells derived from other biological species. In addition, the cells of the present disclosure are suitable for detecting a chemical substance using a plurality of sensor proteins.

[0085] The cells of the present disclosure and the cell chip of the present disclosure can be applied in the detection of chemical substances, particularly odor substances. The chemical substances may, for example, be chemical substances in a test sample of a body fluid (e.g., urine, blood, saliva), air (e.g., air in a room, air in a package), water (e.g., river water, sea water, tap water, up-water, down-water), and the like. In this case, the chemical substances in the test sample can reach the cells and come into contact with the sensor proteins of the cells, for example, by bringing the cells of the present disclosure into contact with the test sample or by adding the test sample to the partitions of the cell chip of the present disclosure. The chemical substances can be detected, for example, by detecting ions flowing into the cells (e.g., by a protein that develops color or emits light by ions).

[0086] Examples

[0087] Hereinafter, the present application will be described in detail based on examples, but the present application is not limited to these examples.

[0088] Test Example 1. Cold storage resistance test 1

[0089] Each of cells derived from Spilosoma imparilis (SpIm cells), Sf9 cells, and HEK293 cells was inoculated at 3 x 10 3 The cell suspension was stained with trypan blue, and the number of cells at the time of inoculation was measured using a countess II FL Automated Cell Counter (Thermo Fisher). In addition, the culture media used were as follows: SpIm cells; Sf-900 III SFM medium, Sf9 cells; Sf-900 III SFM medium, HEK293 cells; DMEM (Dulbecco's Modified Eagle's Medium) (4.5 g / l glucose) (containing L-glutamine, HEPES, not containing pyruvic acid)).

[0090] After the cells were inoculated, the 96-well plates were wrapped with aluminum foil and left to stand in a refrigeration room (4°C). From the start of the standing, the cell survival rate was determined by the MTT test method (kit manufactured by Roche) on day 14, 28, and 35 for SpIm cells, on day 1, 3, 7, and 14 for Sf9 cells, and on day 1, 2, and 5 for HEK293 cells.

[0091] The MTT test was performed according to the procedure attached to the kit as follows. After adding the MTT solution at 10 μL / well, it was left to stand in a 27°C incubator for 4 hours. Subsequently, MTT elution solution was added at 100 μL / well, and left to stand in a 37°C incubator overnight. Then, the absorbance at 560 nm was measured using a TECAN infinite M200 pro. The absorbance of the sample after inoculation was divided by the absorbance of the sample at the time of inoculation (day 0) to calculate the cell survival rate (%).

[0092] The results are shown in Figure 1 It was known that SpIm cells showed higher survival rates when stored for longer periods in a refrigerated state.

[0093] Test Example 2. Cold storage resistance test 2

[0094] <2-1. Preparation of stable expression cells>

[0095] A transposon vector in which an olfactory receptor protein (ORA) coding sequence, an olfactory receptor co-receptor coding sequence, a calcium sensor photoprotein coding sequence, and a puromycin resistance gene coding sequence were arranged under the control of a promoter sequence, and these sequences were arranged between 5' ITR (inverted repeat sequence) and 3' ITR, was introduced into SpIm cells, and a stable olfactory receptor expression SpIm cell (hereinafter, ORA cell) in which the above-described coding sequences and the promoter sequence were integrated into the chromosomal genomic DNA was prepared by selection with puromycin.

[0096] <2-2. Measurement of sensor function>

[0097] ORA cells were inoculated at 1 x 10 5 / 200 μL / well in a 96-well plate CORNING 3909 and left to stand in a 27°C incubator (not supplied with CO2). The culture medium used was Sf-900 III SFM medium. The count of cells was measured using a countess II FL Automated Cell Counter (Thermo Fisher) after staining the cell suspension with trypan blue. After 24 hours from inoculation, the culture solution was removed from the 96-well plate and exchanged for 0.1% BSA HBSS (-) (without phenol red) 80 mL. The fluorescence intensity (GCaMP) was measured using FlexStation3 5 minutes after the liquid exchange. Specifically, compound a (a substance responsive to ORA) adjusted to each concentration (0, 0.01, 0.1, 1, 10, 100 μM) was added to each well, and the change in fluorescence intensity before and after the addition was quantified using a microplate reader (FlexStation3, Molecular Devices).

[0098] <2-3. Measurement of sensor function after cold storage>

[0099] After inoculating ORA cells as in 2-2 above, the cells were left to stand in an incubator at 27°C (without CO2 supply) for 6 hours, and then left to stand in a cold storage room (4°C) with light shielding by aluminum foil. After 72 hours, the fluorescence intensity before and after addition of the compound a was measured as in 2-2 above.

[0100] <2-4. Results>

[0101] The results are shown in Table 2-4. Figure 2 It was known that Splm cells can be used as a chemical substance response sensor, and furthermore, that the sensor function thereof remains stable after cold storage.

[0102] Test Example 3. Evaluation test of chemical substance response activity

[0103] Using Splm cells, stable Splm cells expressing each of the olfactory receptor proteins (ORA, ORB, ORC, ORD, ORE, ORF, AaOR47) were prepared as in 2-1 above. Using each of the cells, the chemical substance response activity was evaluated as in 2-2 above.

[0104] The results are shown in Table 2-5. Figure 3 and Figure 4 It was known that Splm cells also exhibit chemical substance response activity in cases where a plurality of olfactory receptors are used.

Claims

A cell derived from an insect of the family Tetraodonta, comprising an exogenous polynucleotide containing a coding sequence for a sensor protein.

2. The cell according to claim 1, wherein The sensor protein is an olfactory receptor protein.

3. The cell according to claim 1, wherein The sensor protein is an insect olfactory receptor protein.

4. The cell according to claim 1, wherein The exogenous polynucleotide comprises a coding sequence for an olfactory receptor co-receptor protein and a coding sequence for a color-developing or light-emitting protein.

5. The cell according to claim 1, wherein The exogenous polynucleotide comprises a coding sequence of a drug resistance gene.

6. The cell according to claim 1, wherein The exogenous polynucleotide is integrated into the genomic DNA.

7. The cell according to claim 1, wherein Insects of the family Echinops are insects of the genus Echinops.

8. The cell according to claim 7, wherein The insect of the genus Snow Moth is the mulberry-spotted Snow Moth.

9. A cell having cryoresistant properties, comprising an exogenous polynucleotide comprising a coding sequence for a sensor protein.

10. The cell according to claim 9, which is determined to be cryoresistant when subjected to the following cryoresistant test: <Refrigeration resistance test> Step (1): Cells were plated at 1x10 5 / 200μL / partition inoculation, let stand at 27℃ for 6 hours, Step (2): The plate was shielded from light with aluminum foil and allowed to stand at 4°C for 72 hours before measuring the fluorescence intensity. Step (3): After adding the substance that the sensor protein responds to each partition, the fluorescence intensity is measured. Step (4): When the fluorescence intensity measured in step (3) is greater than the fluorescence intensity measured in step (2), the cells are judged to be cryopreservation-resistant. The cell according to claim 9 or 10, wherein the survival rate of the cell after storage at 4°C for 15 days is 50% or more. 12 . A cell chip comprising a partition containing the cells according to claim 1 . The cell chip according to claim 12 , which is used for detecting chemical substances.

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