Cells derived from polypedilum vanderplanki and odor sensor equipped therewith

Abstract

To provide cells that express a membrane protein as an exogenous protein but can be stably transported without a complex culture apparatus.SOLUTION: By using cells derived from Polypedilum vanderplanki expressing an exogenous membrane protein, the membrane protein can be stably transported without a complex culture apparatus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to cells derived from the sleeping chironomid (Polypedilum vanderplanki) that express an exogenous membrane protein, an odor sensor comprising said cells, and a method for detecting odorants using said cells. [Background technology]

[0002] The sleeping chironomid is an insect with extreme desiccation tolerance known as xerosis. It is known that its larvae, even when they have almost completely lost all moisture in their bodies and become desiccated, resume development as if nothing had happened when they were returned to water. Focusing on this characteristic, research on cultured cells derived from the sleeping chironomid has also been conducted. For example, it is known that cultured cells Pv11 can survive and resume proliferation after rehydration even when stored dry at room temperature for over a year (Non-Patent Document 1). It is also known that Pv11 cells maintain the activity of enzymes in their cytoplasm even after prolonged desiccation (Non-Patent Document 2).

[0003] Conventional techniques such as bacterial culture, PCR, chromatography, and gene expression analysis are used to identify the origin of agricultural products and monitor bacterial infection status in the food hygiene field. There is a need for odor sensors that can replace these conventional techniques, allowing for more sensitive and real-time analysis by detecting minute amounts of odorous substances. To date, various odor sensors, such as semiconductor gas sensors, have been developed. However, the current situation is that the only way to detect minute amounts of odorous substances is to rely on dogs' sense of smell (Non-Patent Document 3). Therefore, as a biodevice with an odor detection function equivalent to that of a dog's sense of smell, development is underway to arrange cells that detect specific odors on an array, detect and amplify the cell's response signal to the odor, integrate the signal with AI, etc., and display the odor composition. Such devices are being developed using insect membrane proteins such as olfactory receptor proteins (ORs), olfactory receptor protein co-receptors (Orco) that form cation channels together with ORs, and GCaMP (GFP-based Ca 2+ methyltransferase), a fluorescent marker protein that detects changes in intracellular calcium concentration. 2+ An odor sensor has been developed in which calmodulin protein (Non-Patent Document 4) is expressed in cultured insect cells Sf21, and the cells are arranged in an array to detect specific odor signal patterns for specific odorants (Patent Document 1). Furthermore, development of a CMOS sensor that can detect with high sensitivity minute action potentials in the olfactory nerve that respond to specific odor substances is also underway (Non-Patent Document 5). It is expected that the development of odor biosensors using cultured cells will continue to progress.

[0004] A problem with existing biosensors that use cultured cells or biological tissues has been pointed out: they are short-lived because they rely on biological parts. Furthermore, biosensors require complex culture equipment, making them difficult to transport. When considering using biosensors as odor sensors in the field, the odor sensors must be transported, and they must also be stored in an environment where humidity and temperature are not controlled until they are ready for use. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2013-27376 [Non-patent literature]

[0006] [Non-Patent Document 1] K. Watanabe et al. / Cryobiology 73 (2016) 93-98 [Non-patent document 2] Kikuta et al. "Towards water-free biobanks: long-term dry-preservation at room temperature of desiccation sensitive enzyme luciferase in airdried insect cells" Scientific Reports, 2017 (Published online 26 July 2017) [Non-patent document 3] Tetsuo Nozawa, NIKKEI Electronics, pp. 60-69, June 2015, Nikkei BP [Non-patent document 4] Nature Biotechnology volume 19, pages 137-141 (2001) [Non-Patent Document 5] T. Datta-Chaudhuri et al. / Sensors and Actuators B 235 (2016) 74-78 Summary of the Invention [Problem to be solved by the invention]

[0007] In order to use cells expressing membrane proteins immediately when needed, the cells must be stored in a state in which the extracellular domain, transmembrane domain, and cytoplasmic domain that constitute the membrane protein are expressed and maintained. However, maintaining cells typically requires complex culture equipment, making them difficult to transport over long distances. Furthermore, membrane proteins, particularly their extracellular domains, are generally directly exposed to environmental stresses such as high and low temperatures and dryness, making it difficult to stably maintain membrane proteins in the cell membrane. Therefore, the present invention aims to provide cells that express membrane proteins as exogenous proteins but can be stably transported without complex culture equipment. [Means for solving the problem]

[0008] As a result of intensive research to solve the above-mentioned problems, the inventors discovered that if an exogenous membrane protein is expressed in cells derived from the sleeping chironomid, the cells can be stored at room temperature after drying, and can be easily rehydrated and used when needed, thereby completing the present invention.

[0009] That is, the present invention relates to the following [1] to

[14] . [1] Cells derived from the sleeping chironomid (Polypedilum vanderplanki) expressing at least one foreign membrane protein. [2] The cell according to [1], wherein the exogenous membrane protein is functional even when the cell is rehydrated after drying. [3] The cell according to [1] or [2] above, wherein the exogenous membrane protein comprises an olfactory receptor protein (OR) and / or an olfactory receptor protein co-receptor (Orco). [4] The cell according to any one of [1] to [3] above, wherein the exogenous membrane proteins are OR and Orco. [5] The cell according to any one of [1] to [4] above, wherein the gene for the exogenous membrane protein is operably linked to a 121 promoter. [6] The cell according to any one of [1] to [5] above, which further expresses one or more additional foreign proteins. [7] The cell according to [6], wherein the additional exogenous protein comprises a fluorescent marker protein that detects changes in intracellular calcium concentration. [8] The cell according to any one of [1] to [7] above, wherein the cell is derived from a Pv11 cell. [9] An odor sensor comprising cells derived from the sleeping chironomid expressing OR and Orco.

[10] The odor sensor according to [9], wherein the cells further express a fluorescent marker protein that detects changes in intracellular calcium concentration.

[11] The odor sensor according to [9] or

[10] above, wherein the cells are immobilized on a substrate.

[12] An odor detection method, comprising the step of detecting an odorant using cells derived from the vanderplanki expressing OR and Orco.

[13] An odor detection method comprising the step of detecting an odorant or identifying an odor using a collection of cells derived from Polypedilum vanderplanki expressing one or more ORs and Orco.

[14] A method for preserving an exogenous membrane protein, comprising: introducing a gene encoding the foreign membrane protein into a cell derived from Polypedilum vanderplanki; Culturing the cells to express the exogenous membrane protein in the cell membrane; drying the cells; A method comprising:

[15] The method according to

[14] , further comprising the step of suspending the cells in a solution containing a dry-protectant prior to the drying step.

[16] A method for detecting or identifying an odor, comprising: (a) providing a desiccant-derived biosensor cell expressing one or more ORs and / or Orco; (b) contacting the biosensor cells with at least one test compound or sample; (c) detecting a signal indicative of activation of one or more ORs in the biosensor cells; A method comprising:

[17] A device for detecting the presence of an odorant or for identifying an odor, comprising: A device comprising desiccant-derived biosensor cells expressing one or more ORs that are activated upon contact with an odorant.

[18] The device according to

[17] , which is a portable device.

[19] A kit for detecting an odorant or identifying an odor, comprising: A kit comprising one or more desiccable insect-derived biosensor cells. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide cells derived from the vanderplanki that express exogenous membrane proteins that can be stored at room temperature after drying and can be easily rehydrated for use when needed. Therefore, for example, by using OR and Orco as the exogenous membrane proteins, a portable odor biosensor can be produced that can be used for food hygiene management, food quality evaluation, and the like. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing one embodiment of a cell according to the present invention. [Figure 2] This is a schematic diagram of the genetic organization used to generate the Pv11-GCaMP6f-Orco-OR47a stable cell line (Orco-OR47a stable cell line). The lightning bolt symbol indicates the three bases prior to the PAM sequence (NGG), which is the cleavage site for the CRISPR Cas9 protein. [Figure 3] This is a graph showing the fluorescence intensity when an Orco-OR47a stable expressing cell line was contacted with VUAA1, an Orco agonist (ns: non-significant, ****: p<0.0001). [Figure 4] This is a graph showing the fluorescence intensity when an Orco-OR47a stable expressing cell line was contacted with pentyl acetate, a ligand for OR47a (ns: non-significant, ****: p<0.0001). [Figure 5] This is a graph showing the survival rates of the Pv11 wild-type strain (Pv11), the Pv11-GCaMP6f-Orco stably expressing cell line (Orco stably expressing cell line), and the Orco-OR47a stably expressing cell line one hour after drying and rehydration. [Figure 6A] This is a graph showing the response functions of VUAA1 and pentyl acetate and acetophenone in the Orco-OR47a stably expressing cell line before drought treatment, as measured by changes in fluorescence intensity (**: p<0.01, ***: p<0.001). [Figure 6B]This graph shows the change in fluorescence intensity of the response function of the Orco-OR47a stably expressing cell line to VUAA1 and pentyl acetate one hour after rehydration following desiccation (***: p<0.001, ****: p<0.0001). [Figure 7A] This graph shows the change in fluorescence intensity of the response to VUAA1 in the Orco-OR47a stable cell line 1 hour after desiccation and rehydration. Treatment with CHX (a protein synthesis inhibitor) inhibited de novo protein synthesis of membrane proteins (ns: non-significant). [Figure 7B] This graph shows the change in fluorescence intensity in response to pentyl acetate in the Orco-OR47a stable cell line 1 hour after desiccation and rehydration, and is treated with CHX (a protein synthesis inhibitor) as in Figure 7A (ns: non-significant). [Figure 7C] This graph shows the change in fluorescence intensity in response to VUAA1 in the Orco-OR47a stable cell line 24 hours after desiccation and rehydration, treated with CHX (a protein synthesis inhibitor) as in Figure 7A (****: p<0.0001). [Figure 7D] This graph shows the response function of the Orco-OR47a-expressing cell line 1 hour and 24 hours after rehydration following desiccation treatment, and was treated with CHX (a protein synthesis inhibitor) as in Figure 7A (***: p<0.001). [Figure 8] This is a graph showing the fluorescence intensity when immobilized Orco-OR47a expressing cell lines were contacted with VUAA1 or pentyl acetate (ns: non-significant, *: p<0.05, ****: p<0.01). [Figure 9A] This is a graph showing the change in fluorescence intensity as a function of response to pentyl acetate over time in a ligand assay using a perfusion system with a cell line stably expressing Orco-OR47a. [Figure 9B] 1 is a graph showing the time-dependent response function to pentyl acetate as a change in fluorescence intensity in a ligand assay using a perfusion system with a Pv11-GCaMP6f stably expressing cell line (GCaMP6f stably expressing cell line). [Figure 10] This graph confirms that OR47a expression was optimized by adding untranslated regions (UTRs) to the donor vector (***: p<0.001, ****: p<0.0001). [Figure 11] Photographs of the (a) adult, (b) larval, and (c) desiccated larval stages of the sleeping chironomid. (d) Odorant binding opens the Orco / OR ion channel. Ca2+ influx results in (e) fluorescence of the GCaMP marker. DETAILED DESCRIPTION OF THE INVENTION

[0012] (cell) The present invention relates to cells derived from the S. vanderplanki (Polypedilum vanderplanki) that express an exogenous membrane protein, which are produced by genetic engineering techniques. Cells derived from the S. vanderplanki that can be used to express the exogenous membrane protein include those commonly used in the art, without particular limitation, and include, for example, Pv11 cells and Pv210 cells. The exogenous membrane protein remains functional in the S. vanderplanki-derived cells that express the exogenous membrane protein even after being dried and then rehydrated. The drying conditions are not particularly limited as long as they do not denature the exogenous membrane protein and allow the cells derived from the vanderplanki to function after drying and rehydration. For example, the cells may be dried in an environment with a temperature of 30°C or lower, preferably 25°C or lower, and / or a humidity of 10% or lower, preferably 5% or lower, for 2 days or more, preferably 7 days or more. Drying under such mild conditions allows the exogenous membrane protein to function better after rehydration. From the viewpoint of inducing the expression of an anesthesia-related function, it is preferable to dry the cells derived from the vanderplanki while suspended in a solution containing a drying protectant, such as an insect culture medium containing trehalose or serum.

[0013] Furthermore, "rehydration" refers to returning dried cells to an aqueous solution. The conditions for the rehydration are not particularly limited as long as the dried cells derived from Polypedilum vanderplanki are capable of functioning. For example, a buffer solution containing an insect cell culture medium such as IPL-41 or Dulbecco's phosphate buffer solution (DPBS) may be added to the cells, and a solution containing the insect cell culture medium is preferably added. The cells of the present invention have the characteristic that they can be dried and stored while maintaining their function, even if they contain membrane proteins with extracellular domains, such as functional odorant receptors (heterocomplexes). Even if the dried cells are stored at room temperature for more than 7 days, more than 20% remain viable after rehydration, and even if they are stored at room temperature for more than 372 days, at least 1% remain viable after rehydration.

[0014] Furthermore, the cells express exogenous membrane proteins newly produced in vivo within 24 hours after rehydration after drying. However, even without waiting for the expression of exogenous membrane proteins produced in vivo, membrane proteins that had been preserved in the dried state begin to recover immediately after rehydration after drying, and the cell state can be stabilized within, for example, about 10 minutes. Furthermore, even if the cells are dried again after the rehydration, the function of the membrane protein can be similarly preserved. Without being bound by any particular theory, it is thought that even when cells derived from the sleeping chironomid expressing an exogenous membrane protein are dried, the exogenous membrane protein remains functionally preserved within the cell membrane due to factors such as the vitrification of trehalose and the molecular shielding effect of LEA proteins, and is able to immediately exert its function after rehydration.

[0015] Examples of the exogenous membrane protein include, but are not limited to, cell surface receptor proteins, channels, and transporters that respond to a wide variety of signals from the external environment, such as chemical substances. Examples of the cell surface receptor protein include G protein-coupled receptor (GPCR) proteins, enzyme-coupled receptors, and ion channel-coupled receptors. Examples of the cell surface receptor protein include olfactory receptor proteins (ORs), olfactory receptor protein co-receptors (Orco), and taste receptors and mechanosensory receptors that have structures similar to ORs and Orcos. In addition, the cells may express one type of the exogenous membrane protein, or two or more types of the exogenous membrane proteins may be co-expressed, or a complex may be formed between the co-expressed exogenous membrane proteins. One aspect of the present invention is a cell derived from the vanderplanki expressing an olfactory receptor protein (OR) and / or an insect olfactory receptor protein co-receptor (Orco). The olfactory receptor protein (OR) is a type of G protein-coupled ion channel present in olfactory receptor neurons. In the present invention, ORs may be derived from vertebrates such as mammals or from insects, but are preferably derived from insects due to the simplicity of functioning with just two proteins, OR and Orco, as well as their superior diversity and odor selectivity.

[0016] As described in Patent Document 1, more than 100 insect olfactory receptor proteins (ORs) have been identified from Drosophila melanogaster, Anopheles gambiae, and Bombyx mori. Each OR exhibits highly specific responses to odorants, such as phenethyl alcohol, methyl benzoate, ethyl benzoate, benzyl alcohol, methyl salicylate, benzaldehyde, pentanal, hexanal, E2-hexanal, 2-heptanone, 6-methyl-5-hepten-2-one, and 2-methylphenol (Hallem et al., Cell 125, 143-160, April 7, 2006). These receptor proteins function as ion channel-coupled receptors, and when a target odorant binds to the receptor, ions enter the receptor-expressing cells.

[0017] Insect ORs may be derived from insects, including, but not limited to, economically or medically important species, and more specifically, from insects of the orders Coleoptera, Lepidoptera, Diptera, or Hymenoptera. In one aspect, insect ORs expressed in cells derived from Polypedilum vanderplanki may be derived from Drosophila, and examples of such Drosophila ORs include, but are not limited to, OR1a, OR2a, OR7a, OR9a, OR10a, OR13a, OR19b, OR19a, OR22a, OR22b, OR22c, OR23a, OR24a, OR25a, OR26a, OR27a, OR28a, OR29a, OR30a, OR31a, OR32a, OR33a, OR34a, OR35a, OR36a, OR37a, OR38a, OR39a, OR40a, OR41a, OR42a, OR43a, OR44a, OR45a, OR46a, OR47a, OR48a, OR49a, OR50a, OR51a, OR52a, OR53a, OR54a, OR55a, OR56a, OR57a, OR58a, OR59a, OR60a, OR61a, OR62a, OR63a, OR64a, OR65a, OR66a, OR67a, OR68a, OR69a, OR70a, OR71a, OR72a, OR73a, OR74a, OR75a, OR76a, OR77a, OR78a, OR79a, OR80a, OR81a, OR82a, OR83a, OR84a, OR85a, OR86a, OR87a, OR88a, OR89a, OR90a, OR91a, OR92a, OR93a, Examples include R30a, OR33a, OR33b, OR33c, OR35a, OR42a, OR42b, OR43a, O43b, O45a, O45b, O46a, OR47a, OR47b, OR49a, OR49b, OR56a, OR59a, OR59b, OR59c, OR63a, O65a, O65b, O65c, O67a, O67b, O67c, O67d, and O69a. OR74a, OR82a, OR83a, OR83c, OR85a, OR85b, OR85c, OR85d, OR85e, OR85f, OR88a, OR92a, OR94a, OR94b, OR98a, and OR98b.

[0018] As non-limiting examples, OR85b of Drosophila melanogaster responds to ammonia, a highly hazardous substance; OR46a of Drosophila melanogaster specifically responds to 4-methylphenol present in human sweat; and OR98a of Drosophila melanogaster can detect phenylacetone, a precursor of amphetamine. These ORs can be expressed in cells derived from the midge, S. vanderplanki. The database DoOR2.0 provides insights into the function of Drosophila ORs and quantitative data on in vivo odor responses (D. Munch and C.G. Galizia, "DoOR 2.0 - Comprehensive mapping of Drosophila melanogaster odorant responses," Sci. Rep., 6 (1), 21841 (2016), 10.1038 / srep21841).

[0019] The olfactory receptor protein co-receptor (Orco) is a co-factor for ORs, and when an odorant binds to an OR, it forms a heterocomplex with Orco, which functions as a ligand-gated ion channel. When an odorant binds to ORs, the pore formed by ORs and Orco opens, allowing cations to flow into the cell, generating an action potential. The insect olfactory receptor protein co-receptor (Orco) has a high degree of sequence conservation across species, so Orco can be used not only in Drosophila melanogaster but also in species more suitable for Pv11 cells, such as the sleeping chironomid.

[0020] Furthermore, it is preferable that the cells derived from the vanderplanki according to the present invention have a gene encoding a foreign membrane protein integrated into the genome of the cells, for reasons such as enabling stable expression of the foreign membrane protein. In cells derived from the vanderplanki that express the foreign membrane protein of the present invention, the gene encoding the foreign membrane protein is preferably operably linked to the 121 promoter (SEQ ID NO: 1). The 121 promoter is a strong promoter discovered in the genome of the vanderplanki, and is known to have an ability to produce proteins approximately 1,500 times higher than promoters included in commercially available kits for insect cells. The 121 promoter derived from the vanderplanki has the following sequence shown in SEQ ID NO: 1.

[0021] TIFF2025138913000001.tif133156

[0022] In another aspect of the present invention, cells derived from Polypedilum vanderplanki expressing the foreign membrane protein of the present invention may further express another foreign protein other than the foreign membrane protein. The foreign protein is not particularly limited. For example, if the foreign membrane protein is a G protein-coupled ion channel, the foreign protein may be a fluorescent marker protein capable of detecting ions that enter the cell when the ion channel binds to a ligand. If the function of the foreign membrane protein results in a change in intracellular calcium ions, the foreign protein may be a protein that emits fluorescence in response to calcium ions in the cytoplasm, such as G-CaMP; G-CaMP1.6; GCaMP2; GCaMP3; G-CaMP4.1; GCaMP5; G-CaMP6,7,8; GCaMP6f,6m,6s; jGCaMP7f,7s,7b,7c; and GCaMP-X.

[0023] One embodiment of the cells derived from the vanderplanki of the present invention is described with reference to FIG. 1. The cells of one embodiment of the present invention are Pv11 cells that co-express OR and Orco as exogenous membrane proteins and express exogenous GCaMP intracellularly. Even if the cells are dried, they can be restored to functional cells by rehydration, and the hydrated and dried states are reversible. The dried cells can be stored at room temperature, making them portable without the need for complex culture equipment. Furthermore, the exogenous membrane protein remains functional even after the cells are dried and rehydrated. Specifically, OR and Orco form an odorant receptor complex in the cell membrane. When the complex comes into contact with an odorant that specifically reacts with the complex, the calcium ion concentration in the cytoplasm increases in response. This response can be reproduced within approximately one hour even after the cells are dried and rehydrated. Furthermore, GCaMP emits a fluorescent signal in response to calcium ions, allowing the increase in calcium ion concentration due to the response to the complex to be visualized. Furthermore, 24 hours after rehydration, the cell's response to odorants was strongly enhanced by the de novo synthesized Orco and OR proteins. Thus, Pv11 cells expressing OR, Orco, and GCaMP can exhibit the function of the membrane protein odorant receptor complex when hydrated, even after repeated hydration and desiccation. Therefore, they can be transported in a dry state and rehydrated when needed to detect specific odorants.

[0024] (Cell production method) A gene encoding a foreign membrane protein can be obtained by, for example, extracting mRNA from the foreign membrane protein using a conventional method, synthesizing and isolating cDNA, and then performing PCR using the cDNA as a template and appropriate PCR primers to obtain a portion of the gene. Similarly, a gene encoding an additional foreign protein can be obtained by PCR using cDNA as a template and appropriate PCR primers. The foreign membrane protein can also be cloned using other conventional methods. The foreign membrane protein and / or additional foreign protein can be incorporated into an expression vector, and cells derived from Polypedilum vanderplanki can be transformed with the expression vector to prepare cells that express the protein of interest. There are no particular limitations on the foreign protein expression vector, as long as it is replicable in the host cell.

[0025] To express OR and / or Orco, recombinant DNA techniques well known in the art can be used. Expression vectors are not particularly limited, and nucleic acids in the form of plasmids, cosmids, phagemids, phages, or viral vectors may be used. For construction of expression vectors using recombinant DNA techniques, reference may be made to Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, (2001)), FM Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994)), and Marston, F. (1987) DNA Cloning Techniques.

[0026] In one aspect, the expression vector may include a regulatory sequence that is operably linked to the nucleic acid and thereby affects the transcription and / or translation of the nucleic acid encoding OR and / or Orco. Such regulatory sequences may include, but are not limited to, a promoter sequence (e.g., 121 promoter) or a transcription termination sequence (e.g., polyadenylation signal). As used herein, "operably linked" refers to a linkage that affects the transcription and / or translation of the nucleic acid. The regulatory sequence may also include an enhancer sequence that functions to regulate the transcription of the nucleic acid. In one embodiment, the expression vector may further comprise a selectable marker gene, which encodes a trait that allows for the selection of a host microorganism containing the marker gene, and which may generally be an antibiotic resistance gene. In certain aspects, the expression vector may contain restriction enzyme recognition sites for easy cloning of nucleic acids encoding OR and / or Orco.

[0027] Furthermore, to obtain a stable expression strain expressing the foreign membrane protein and / or the additional foreign protein, a gene encoding the foreign membrane protein and / or the additional foreign protein may be knocked into a cell derived from Polypedilum vanderplanki by applying a known genome editing technique using ZFN, TALEN, CRISPR / Cas9, etc. In the present invention, among known genome editing techniques, the CRIS-PITCh method (Nakade S et al., "Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR / Cas9," Nature Communications 5:5560 2014) is preferred for knock-in because of its ease of operation and high accuracy of gene insertion. The CRIS-PITCh method is a gene knock-in method that utilizes the repair pathway MMEJ (non-homologous end joining) by introducing into cells a donor vector containing a cassette in which short homologous sequences (microhomologies) of approximately 20 base pairs are added to both ends of a foreign gene, and simultaneously cutting the genomic region of the target gene and both outside of the foreign gene. Alternatively, sequences encoding multiple proteins of interest may be incorporated into a single expression vector or donor vector, or each sequence encoding a protein of interest may be incorporated into a separate vector. Expression of the foreign membrane protein and / or additional foreign protein may be optimized in the expression vector or donor vector. Such optimization may be achieved by, but is not limited to, adding an untranslated region (UTR), codon optimization, or adding a cell membrane transport signal peptide. Preferably, expression of the foreign membrane protein may be optimized by adding a UTR. Furthermore, in a cell derived from Polypedilum vanderplanki expressing the foreign membrane protein of the present invention and / or an additional foreign protein, the gene encoding the foreign protein is preferably designed to be operably linked to the 121 promoter (SEQ ID NO: 1).

[0028] (Odor sensor) The present invention also relates to an odor sensor comprising cells derived from the vanderpig chironomid expressing at least an OR, preferably co-expressing an OR and Orco. The cells may further express, as the additional foreign protein, a fluorescent marker protein that detects changes in intracellular calcium concentration. Alternatively, instead of expressing a fluorescent marker protein in the cells, a calcium ion-dependent fluorescent dye, such as Calbryte, may be added to the cells. TM 520AM and Calbryte TM 590AM, Calbryte TM Alternatively, a low molecular weight fluorescent substance such as Fluo-630AM, Fluo-5F AM, Fluo-4 AM, Fluo-4FF AM, Fluo-3 AM, Fura-2 AM, Indo-1 AM, Rhod-2 AM, Rhod-3 AM, X-Rhod-1 AM, X-Rhod-5F AM, or Oregon Green (registered trademark) 488 BAPTA series may be added to the cell membrane to enable monitoring of intracellular calcium ion concentrations by changes in fluorescence. In the odor sensor, it is preferable that the fluorescence intensity after contact with an odorant to which the OR specifically reacts increases by at least 1%, preferably at least 5%, compared to the fluorescence intensity before contact. Furthermore, it is preferable that the odor sensor comprises the above-mentioned stably expressing cell line.

[0029] In one embodiment, the cells are immobilized on a substrate (such as a chip). The means for immobilizing the cells is not particularly limited, but for example, the cells may be immobilized on the substrate using a cell membrane modifying agent (BAM; Biocompatible Anchor for cell Membrane) or polyethylenimine, which is an anchoring system that utilizes electrostatic interaction. In addition, the odor sensor is configured such that the cells are immobilized on the substrate at a density of at least 1 × 10 4 pieces / cm 2 , preferably at least 1 x 10 5 Preferably, the odor sensor comprises cells derived from the vanderplanki. By providing multiple types of spots formed by immobilizing cells for each type of OR on the substrate, an array capable of simultaneously detecting different types of odorants can be produced. When the array is contacted with a test sample of unknown composition, if the test sample contains an odorant to which any of the ORs specifically responds, fluorescence is observed at the spot where the cells expressing that OR are immobilized. Furthermore, the odor sensor can be stored in a dry state, and can be immediately used for odor determination by rehydrating the cells at the time of use. The methods for drying and rehydrating the cells, and the methods for detecting and analyzing the fluorescent signals can be any methods commonly used in the art, without particular limitations. The odor sensor can be applied, for example, to the management of prohibited imports at airports and in the medical field, where odors are used to detect cancer early. It can also be used in the agricultural field, for example, in food hygiene management and food quality evaluation based on the detection of faint odors (e.g., geosmin produced by mold). Another application example is the early detection of agricultural pests by detecting specific odor patterns.

[0030] (Odor detection method) The present invention also relates to an odor detection method comprising the step of detecting an odorant or distinguishing an odor using cells derived from the vanderplanki expressing ORs and Orco. The present invention also relates to an odor detection method comprising the step of detecting an odorant or distinguishing an odor using a collection of cells derived from the vanderplanki expressing one or more ORs and Orco.

[0031] Alternatively, the present invention relates to a method for detecting or identifying an odor, the method comprising: (a) providing a desiccant-derived biosensor cell expressing one or more ORs and / or Orco; (b) contacting the biosensor cells with at least one test compound or sample; (c) detecting a signal indicative of activation of one or more ORs in the biosensor cells; As the insect-derived biosensor cells that can be dried, the cells derived from the vanderplanki described above as one aspect of the present invention can be used.

[0032] (Devices and Kits) The present invention also relates to a device for detecting the presence of an odorant or for distinguishing odors, the device including a desiccable insect-derived biosensor cell expressing one or more ORs that are activated upon contact with the odorant. In one embodiment, the device may be a portable device or the like. The present invention also relates to a kit for detecting an odorant or for distinguishing odors, the kit including one or more desiccable insect-derived biosensor cells. The desiccable insect-derived biosensor cell may be a polypedilum vanderplanki cell, as described above as one embodiment of the present invention.

[0033] (Methods for preserving exogenous membrane proteins) The present invention also relates to a method for preserving an exogenous membrane protein in a dry state. The method includes the steps of introducing a gene encoding the exogenous membrane protein into cells derived from Polypedilum vanderplanki, culturing the cells to express the exogenous membrane protein in the cell membrane, and drying the cells. According to this method, the membrane protein preserved in a dry state begins to recover immediately after the cells expressing the exogenous membrane protein are rehydrated after drying, and the cell state can be stabilized within, for example, about 10 minutes. Furthermore, from the perspective of inducing the expression of an anesthesia-related function, it is preferable to further include a step of suspending the cells in a solution containing a dry protection agent, such as trehalose or serum-containing insect culture medium, prior to the drying step.

[0034] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0035] 1. Preparation of Transient Expression Strains GCaMP6f cDNA was amplified by PCR using standard methods, and the amplified DNA sequence encoding GCaMP6f (SEQ ID NO: 2) was inserted downstream of the 121 promoter of the pPv121-MCS vector (Tokumoto et al., doi: https: / / doi.org / 10.1101 / 2020.05.29.123570 "Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11") to create the pPv121-GCaMP6f vector. Similarly, Drosophila melanogaster Orco cDNA was amplified by PCR using standard methods, and the amplified DNA sequence encoding Orco (SEQ ID NO: 3) was inserted downstream of the 121 promoter of the pPv121-MCS vector to create the pPv121-Orco vector. In addition, the OR47a cDNA derived from Drosophila melanogaster was amplified by PCR using standard methods, and the amplified DNA sequence encoding OR47a (SEQ ID NO: 4) was inserted downstream of the 121 promoter in the pPv121-MCS vector to create the pPv121-OR47a vector. In each expression vector, each inserted gene is operably linked to the 121 promoter.

[0036] The resulting pPv121-GCaMP6f, pPv121-Orco, and pPv121-OR47a vectors were co-transfected into Pv11 cells (see Miyata Y., et. al, Scientific Reports, 9(1), 7004.(2019)) to generate Pv11 transient expression strains that transiently express GCaMP6f, Orco, and OR47a proteins. If the membrane proteins Orco and OR47a are expressed and GCaMP6f is expressed in the cytoplasm, stimulation of Pv11 transient-expressing cells with Orco or OR47a agonists or ligands induces calcium ion influx into the cells, resulting in the observation of GCaMP6f fluorescence. Therefore, dimethyl sulfoxide (DMSO; negative control), VUAA1 (an Orco agonist), and pentyl acetate (an OR47a ligand) were added to the culture medium containing the resulting Pv11 transient-expressing cells, and the cells were photographed under a fluorescence microscope to observe fluorescence. Fluorescence was observed both with VUAA1 and pentyl acetate stimulation, confirming the expression of each foreign protein in the resulting Pv11 cells. Therefore, we confirmed that Pv11 cells can express each foreign membrane protein at least transiently and that it is functional.

[0037] TIFF2025138913000002.tif133156

[0038] TIFF2025138913000003.tif143156

[0039] TIFF2025138913000004.tif117156

[0040] 2. Creation of cell lines stably expressing exogenous membrane proteins and / or additional exogenous proteins (1) Method for generating a stable GCaMP6f-expressing cell line The Pv.00443 gene, located downstream of the 121 promoter in Pv11 cells, was used as the target gene. A gene fragment containing the GCaMP6f DNA sequence with a P2A (self-cleaving 2A peptide) sequence added immediately before the stop codon of the Pv.00443 gene was knocked in using the CRIS-PITCh method (Nakade S et al., "Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR / Cas9," Nature Communications 5:5560 2014). In order to efficiently obtain knock-in transformants, we also added a DNA sequence encoding P2A to the zeocin-resistant enzyme (Zeo r The sequence encoding the gene was also knocked in at the same time. We then established a cell line stably expressing Pv11-GCaMP6f by performing single-cell sorting using Zeocin selection and GCaMP6f fluorescence.

[0041] (2) Method for generating stable Orco-expressing cell lines As in 2.(1) above, an exogenous gene fragment consisting of the DNA sequence of GCaMP6f with a P2A sequence attached and the DNA sequence of Orco with a P2A sequence attached was knocked in immediately before the stop codon of the Pv.00443 gene downstream of the 121 promoter using the CRIS-PITCh method, and an Orco-expressing cell line was established by single-cell sorting using the fluorescence of GCaMP6f.

[0042] (3) Method for generating stable Orco-OR47a expressing cell lines As in 2.(1) above, a foreign gene fragment consisting of the DNA sequence of GCaMP6f with a P2A sequence added and the DNA sequence of Orco with a P2A sequence added immediately before the stop codon on separate alleles of the Pv.00443 gene downstream of the 121 promoter, and a foreign gene fragment consisting of the DNA sequence of Zeo with a P2A sequence added rForeign gene fragments consisting of the OR47a DNA sequence and the OR47a DNA sequence with the P2A sequence added were knocked in using the CRIS-PITCh method, and Orco-OR47a-expressing cell lines were established by selecting with Zeocin and performing single-cell sorting using the fluorescence of GCaMP6f. Figure 2 shows the genetic organization of the Orco-OR47a stable cell line. The lightning bolt symbol indicates the cleavage site.

[0043] 3. Evaluation of stable expressing cell lines (1) Knock-in check To confirm whether the target gene had been knocked into the target site in the established stable-expressing cell lines, genomic PCR was performed upstream and downstream of the Pv.00443 gene, and a band of the expected size was detected. After gel purification of this band, DNA sequencing confirmed that the sequence matched the target (foreign) gene. From these results, it was confirmed that the target gene had been successfully knocked into the target site in each of the established cell lines.

[0044] (2) Functional confirmation of foreign proteins in GCaMP6f stable expressing cell lines The functionality of GCaMP6f in the resulting cell lines was evaluated using ionomycin (Fujifilm Wako Pure Chemical Industries), a calcium ion permeant. Ionomycin was dissolved in dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical Industries) and added to the cell suspension at a final concentration of 1% DMSO and 1 μM in IP1-41 medium. As a negative control, IP1-41 medium was also added to the cell suspension at a final concentration of 1% DMSO. Brightfield and fluorescence images (ex: 470 / 40 nm; em: 525 / 50 nm) of the cells were taken before and after addition of the solution (all-in-one fluorescence microscope BZ-X710, Keyence). In the stable GCaMP6f-expressing cell line, the addition of DMSO did not result in an increase in fluorescence, whereas the addition of ionomycin resulted in a stronger fluorescence than before. This confirmed that the expressed GCaMP6f was functional in the stable GCaMP6f-expressing cell line. Statistical analysis of the fluorescence intensity was performed using Prism8 (GraphPad) with one-way ANOVA, followed by Tukey's multiple comparison test as a post-hoc test.

[0045] (3) Functional confirmation of exogenous membrane proteins in Orco-OR47a stable expressing cell lines The amount of fluorescence expression was confirmed using the same procedure as in 3.(2) above. Instead of ionomycin, a solution of VUAA1, an Orco agonist, was added to the cell solution at a final concentration of 100 μM. Similarly, a solution of pentyl acetate, an OR47a ligand, was added to the cell solution at a final concentration of 1 mM. Fluorescence images were taken at excitation / emission wavelengths of 488 nm / 525 nm. Fluorescence intensity was quantified by surrounding the cells in the captured images and measuring the fluorescence intensity (F) over time. The results are shown in Figures 3 and 4. As shown in Figure 3, the Orco-OR47a stable cell line did not respond to a control solution containing DMSO (negative control), but showed an increase in fluorescence intensity in response to VUAA1, indicating functional expression of Orco. Furthermore, as shown in Figure 4, an increase in fluorescence intensity was observed in response to pentyl acetate, indicating that OR47a was functionally expressed. Therefore, it was found that both Orco and OR47a functioned normally in the stable cell lines we created. Statistical analysis was performed as described in 3.(2) above.

[0046] (4) Cell viability assay of GCaMP6f stable expressing cell lines To evaluate the desiccation tolerance of the cell lines, the cells were dried using the following protocol. 4×107 The Pv11-GCaMP6f stable expressing cell line was centrifuged at 700g for 3 minutes, and the supernatant was removed. The precipitated cells were suspended in a 600mM trehalose mix (a 9:1 mixture of 600mM trehalose and IPL-41 medium), transferred to a 2ml flask, and incubated at 25°C for 48 hours. After 48 hours, the resulting cells were centrifuged at 700g for 3 minutes, and the supernatant was removed. The precipitated cells were suspended in 400µl of the 600mM trehalose mix, and 40µl of the suspension was dropped onto a 35mm dish. The 35mm dish was then dried at 25°C and humidity of 10% or less for 10 days.

[0047] Cells were rehydrated by adding 1 ml of IPL-41 medium after 10 days of desiccation. For the cell viability assay, a cell staining solution mix was prepared by mixing 50 μl of IPL-41 medium with 1.5 μl of PI solution (Dojindo Laboratories) and 4 μl of Hoechst 33342 solution (Dojindo Laboratories). 2 μl of the cell staining mix and 40 μl of cells 1 hour after rehydration were placed in a 1.5 ml tube and double-stained. Images were taken using PI (ex: 545 nm / em: 605 nm) and Hoechst (ex: 405 nm / em: 460 nm). The resuscitation rate was calculated by dividing the number of live cells (Hoechst+, PI-) in the image by the total number of cells (Hoechst+). One hour after rehydration, the viability of control Pv11 cells was 13%, while that of the GCaMP6f stable cell line exceeded 20%. While the difference in viability could be explained by the influence of genetic background, this study demonstrated that at least the GCaMP6f stable cell line maintained desiccation tolerance.

[0048] (5) Cell viability assay of Orco stable expressing cell lines and Orco-OR47a stable expressing cell lines The Pv11 wild-type strain, the Orco stably expressing cell line, and the Orco-OR47a stably expressing cell line were desiccated under the conditions described in 3.(4) above, rehydrated, and their viability was confirmed one hour later. The results are shown in Figure 5. Although there were differences in survival rates due to the influence of genetic background, it was confirmed that both the Orco stably expressing cell line and the Orco-OR47a stably expressing cell line maintained desiccation tolerance.

[0049] (6) Evaluation of the function of exogenous membrane proteins after drying and rehydration To evaluate the function of exogenous membrane proteins, we measured the fluorescence intensity in response to stimulation with VUAA1 (an Orco agonist) or pentyl acetate (an OR47a ligand) using stable Orco and stable Orco-OR47a cell lines before desiccation and 1 hour after desiccation and rehydration, as described in 3.(2) above. Fluorescence intensity was quantified from photographs and calculated as ΔF / F0 using the following formula:

number

[0050] Specifically, the fluorescence image was taken under the condition of excitation wavelength / fluorescence wavelength of 488 nm / 525 nm, and the fluorescence intensity was quantified by surrounding a specific cell in the image and measuring the fluorescence intensity (F i,after The initial value of the fluorescence intensity (F i,0 ) and the change in fluorescence (ΔF i ) and calculate the initial fluorescence intensity (F i,0 ) and normalized by dividing by (ΔF i / F i,0 ) Select n cells from the captured image and measure ΔF i / F i,0 The results were averaged (with SD) and plotted as a graph. For example, 25 cells were selected from the captured images, and the ΔF i / F i,0The results were then averaged (with SD) and plotted as a graph. Figure 6A shows the results for the Orco-OR47a stable expressing cell line before desiccation, and Figure 6B shows the results for the Orco-OR47a stable expressing cell line 1 hour after desiccation and rehydration. Significant differences were observed between the Orco-OR47a stable expressing cell line before desiccation: it responded to the Orco agonist (VUAA1) and pentyl acetate (PA), a specific ligand for OR47a, but not to acetophenone, which is not a ligand for either Orco or OR47a (Figure 6A). Furthermore, in the Orco-OR47a stably expressing cell line, 1 hour after rehydration of the dried cells following desiccation, significant differences in responsiveness to VUAA1 and pentyl acetate were observed compared to the control DMSO (Figure 6B).

[0051] Furthermore, one hour after desiccation and rehydration, VUAA1 stimulation induced fluorescence in both the Orco-OR47a stable cell line and the Orco stable cell line. One hour after desiccation and rehydration, pentyl acetate stimulation induced fluorescence in the Orco-OR47a stable cell line. These results demonstrate that both Orco and OR47a functions are maintained in cells rehydrated after desiccation. As described above, we were able to confirm that the functionality of multiple exogenous membrane proteins was maintained after rehydration, suggesting that various exogenous membrane proteins can be stably stored in a dry state by using cells derived from the vanderplanki as hosts. Furthermore, we found that the exogenous membrane proteins were able to exert their functions within approximately one hour after rehydration.

[0052] (7) Confirmation that the response of exogenous membrane proteins in the Orco-OR47a stable cell line immediately after rehydration is not due to de novo protein synthesis. To confirm that the membrane protein response of the Orco-OR47a stable cell line immediately after rehydration was not due to de novo protein synthesis, the cells were treated with cycloheximide (CHX, a protein synthesis inhibitor). Previous studies have reported that 0.35 mM cycloheximide can inhibit protein production in Pv11 cells. Therefore, in this experiment, cycloheximide was dissolved in IPL-41 medium to a concentration of 0.35 mM. In addition, IPL-41 medium was prepared with cycloheximide at 0.01 mM, 0.07 mM, and 0.7 mM concentrations. Dried cells were rehydrated using 1 ml of cycloheximide-containing IPL-41 medium. The same procedure as in 3.(6) above was repeated 1 hour and 24 hours after rehydration, and fluorescence intensity was measured and analyzed. ΔF / F0 was as described above. The results are shown in Figures 7A and 7B. Figures 7A and 7B are graphs showing the changes in fluorescence intensity of the Orco-OR47a stably expressing cell line to VUAA1 and pentyl acetate, respectively, 1 hour after desiccation and rehydration. Figures 7C and 7D are graphs showing the response function of the Orco-OR47a stably expressing cell line to VUAA1 and pentyl acetate, respectively, as changes in fluorescence intensity 24 hours after desiccation and rehydration.

[0053] One hour after desiccation and rehydration, Pv11 cells showed specific responses to VUAA1 (Fig. 7A) and pentyl acetate (Fig. 7B). The results in Fig. 7A and Fig. 7B showed no significant difference in the function of exogenous membrane proteins depending on the presence or concentration of cycloheximide, indicating that at least a portion of the exogenous membrane proteins Orco and OR47a remained functional on the cell membrane even after the stresses of desiccation, storage, and rehydration. Furthermore, 24 hours after rehydration, the responses of Pv11 cells to VUAA1 (Fig. 7C) and pentyl acetate (Fig. 7D) were strongly enhanced due to de novo synthesis of Orco and OR47a proteins, whereas the responses to exogenous membrane proteins were weak in CHX-treated cells. As a result, we found that Pv11 cells retained some degree of Orco-OR47a function on their plasma membranes even after desiccation and rehydration, independent of de novo protein synthesis.

[0054] 4. Confirmation of the behavior of immobilized Pv11 cells (1) Pv11 cell immobilization To anchor the stable cell lines obtained above, we used a cell membrane modifying agent (BAM: Biocompatible Anchor for Cell Membrane). BAM consists of hydrophilic PEG chains, NHS groups, and hydrophobic oleyl groups. When BAM was applied to a collagen-coated dish, the NHS groups bound to the collagen, and the oleyl groups hydrophobically interacted with the cell membrane, allowing cells to anchor. BAM (SUNBRIGHT OE-040CS, Yuka Sangyo Co., Ltd.) was dissolved in DMSO (Fujifilm Wako Pure Chemical Industries) to a concentration of 10 mM. The 10 mM BAM solution was adjusted to a final concentration of 100 μM in DPBS(-) (Thermo Fisher), and 100 μl of it was added dropwise to a collagen I-coated 48-well microplate (IWAKI) and incubated at 37°C for 1 hour. The BAM solution was then removed, washed once with DPBS(-) (Thermo Fisher) and five times with MilliQ, and allowed to stand to evaporate. The Pv11 cell suspension was centrifuged at 700 g for 3 minutes, the supernatant was removed, and the cell pellet was suspended in DPBS(-) (Thermo Fisher). This was then added dropwise to a BAM-coated dish. After leaving the dish to stand for 15 minutes, DPBS (Ca 2+ , Mg 2+ ) (Thermo Fisher) twice to remove floating cells. The BAM fixation method enabled cells to be immobilized on the plate, and it was confirmed that the cells remained firmly attached even after washing the plate with buffer.

[0055] (2) Functional confirmation of exogenous membrane proteins in immobilized cells Orco-OR47a stable expressing cell lines and Orco stable expressing cell lines were immobilized using the method described in 4.(1) above, and their response to odorants was examined. Each cell was reacted with each substance using the same method as described in 3.(2) above, and the fluorescence intensity was measured. The results for the Pv11-GCaMP6f-Orco-OR47a stable expressing cell line are shown in Figure 8. We also confirmed that the immobilized Pv11-GCaMP6f-Orco-OR47a stable expressing cell line responded to both the Orco agonist VUAA1 and the odorant pentyl acetate. On the other hand, the Pv11-GCaMP6f-Orco stable cell line did not respond to pentyl acetate, but did respond to VUAA1, an Orco agonist.

[0056] 5. Other test examples (1) Ligand assay Ligand assays were performed using a perfusion system with the Orco-OR47a stable expressing cell line and the GCaMP6f stable expressing cell line. Figure 9A shows the results for the Orco-OR47a stable expressing cell line, and Figure 9B shows the results for the Pv11-GCaMP6f stable expressing cell line.

[0057] Orco-OR47a stable cell lines and GCaMP6f stable cell lines were fixed with BAM as described in 4.(1) above and perfused at a flow rate of 1 mL / min using a peristaltic pump (SJ-1211 II-L2, ATTO). The perfusion solution was ACSF buffer (125 mM NaCl, 5.6 mM KCl, 1.25 mM NaHPO, 10 mM HEPES, 1.5 mM CaCl, pH 7.4) as the standard solution. Pentyl acetate was diluted to a final concentration of 1 mM as the OR47a ligand, and DMSO was diluted to 1% by mass as the negative control. The standard solution was perfused, and then switched to 1 mM pentyl acetate or 1% by mass DMSO solution, and perfusion was continued for 5 minutes. Imaging of the cells began approximately 60 seconds after the start of imaging, allowing the 1 mM pentyl acetate or 1% by mass DMSO solution to reach the cells. Bright-field and fluorescent images of the perfused Pv11 cells were taken 30 times at 10-second intervals (LSM700, ZEISS). ΔF / F0 was calculated as described above. 25 cells were selected from the images taken, and ΔF i / F i,0 The results were averaged (with SD) and graphed. These results confirmed that the cell line of the present invention responded immediately after contact with an odorant, and the response continued for up to about four minutes, making it possible to use it as an odor sensor capable of quickly detecting odors.

[0058] 6. An example of optimizing OR47a expression (1) Construction of HiBiT-containing expression vector An expression vector, pPv121-OR47a-linker-HiBiT vector, was prepared by adding a linker sequence (sequence number 5) and a HiBiT tag to the C-terminus of the OR47a-encoding sequence in the pPv121-OR47a vector prepared in 1 above. TIFF2025138913000006.tif16156

[0059] (2) Addition of untranslated regions (UTRs) Two UTRs, Tret1 (Tret1_5'UTR (SEQ ID NO: 6); Tret1_3'UTR (SEQ ID NO: 7)) and g5495 (g5495_5'UTR (SEQ ID NO: 8); g5495_3'UTR (SEQ ID NO: 9)), were cloned using standard methods. By adding these UTRs to the pPv121-Orco vector prepared in 1 above and the pPv121-OR47a-linker-HiBiT vector, pPv121-Tret1-UTR-Orco, pPv121-g5495-UTR-Orco, pPv121-Tret1-UTR-OR47a-linker-HiBiT, pPv121-g5495-UTR-OR47a-linker-HiBiT was produced.

[0060] TIFF2025138913000007.tif75156

[0061] TIFF2025138913000008.tif75156

[0062] TIFF2025138913000009.tif32156

[0063] TIFF2025138913000010.tif90156

[0064] (3) Comparison of OR47a expression levels The expression vectors prepared in 6.(2) above were transfected into Pv11 cells as follows to transiently express each protein. 1: Blank test (no vector introduced) 2: pPv121-Orco vector and pPv121-OR47a-linker-HiBiT 3: pPv121-Tret1-UTR-Orco and pPv121-Tret1-UTR-OR47a-linker-HiBiT 4: pPv121-g5495-UTR-Orco and pPv121-g5495-UTR-OR47a-linker-HiBiT At the same time, as a transfection efficiency control, pPv121(632bp)-Luc2 was transfected into Pv11 cells to express Luc2. For the above 1 to 4, OR47a expressed on the cell membrane was quantified using the HiBiT Extracellular detection system (Promega), and the expression level of Luc2 expressed within the cells was quantified separately using the Nano-Glo (registered trademark) Dual-luciferase (registered trademark) Reporter Assay System (Promega). The expression level of OR47a was corrected for transfection efficiency by dividing the OR47a value quantified by the HiBiT system by the value of Luc2, the transfection control, and is shown in Figure 10. Statistical analysis was performed in the same manner as in 3.(2) above. The results in Figure 10 show that the expression level of OR47a was improved when Tret1-UTR and g5495-UTR were added.

[0065] 7. Supplementary explanation: An example of modified Pv11 cells The sleeping chironomid, an insect that inhabits semiarid regions (Figure 11a), can undergo an anhydrobiosis (xerodialysis) during the larval stage even when completely desiccated (Figures 11b and 11c). The sleeping chironomid possesses unique genes that are expressed during desiccation (MidgeBase, a genome database for the sleeping chironomid (http: / / bertone.nises-f.affrc.go.jp / midgebase / )). Trehalose replaces water molecules lost during the drying process, protecting membranes and proteins from denaturation during dehydration. Other biomolecules, such as LEA (Late Embryogenesis Abundant) proteins, antioxidants, protein repair molecules, and aquaporins, are also important in anhydrobiosis. Larvae in the desiccated state can withstand harsh conditions such as high and low temperatures, vacuum, and radiation. Cultured cells from the sleeping chironomid, the Pv11 strain, can resume cell proliferation upon rehydration, even after pre-treatment with trehalose and subsequent desiccation. Trehalose not only functions as a desiccation protectant but also induces the expression of anhydrobiosis-related genes, so that cultured cells Pv11 derived from the sleeping chironomid Pvmak are easily able to withstand desiccation by pre-treating them with trehalose. If Pv11 cells are cultured in a normal culture medium and released into the environment, they will not be able to continue to grow, so there is no risk of environmental damage even if they are accidentally released.

[0066] Pv11 cells do not require special atmospheric conditions (e.g., CO2) and can be cultured at room temperature (around 25°C). This contrasts with mammalian cells, which require 37°C. This makes Pv11 cells an excellent candidate for sensor development. Furthermore, in a dried state, they can be stored at room temperature for over a year. Even after weeks of storage or transportation at different temperatures during international transport, Pv11 cells can be rehydrated and resume culture. Dried Pv11 cells can be resuscitated after one minute in a 500W microwave oven. These characteristics are unique among potential biohybrid sensor systems. Therefore, sensors based on Pv11 cells, a biological material, can be handled in the same way as conventional polymer-based sensors. Upon rehydration, a significant proportion of OR-expressing cells resumed function and proliferation, even after three years of storage at room temperature. Pv11 cells preserve the activity of cytoplasmic enzymes and the function of membrane proteins. Furthermore, the function of several exogenous proteins has been shown to be maintained even after rehydration. Thus, cell lines stably expressing Orco-Or47a respond to odorants within one hour of rehydration. The odorant response of rehydrated cells is further enhanced by the intracellular synthesis of Orco and ORs over the course of 24 hours after rehydration. More than 900 insect cell lines have already been established, and gene expression systems using these insect cell lines are being used for commercial vaccine production. Screening systems using these insect cells are also being used for the development of new insecticides. The establishment of a heterologous gene expression system capable of producing large amounts of target proteins in Pv11 cells has made it possible to create such odor-sensitive, desiccation-resistant cells. Gene editing technology can be used to express foreign genes without adversely affecting the desiccation tolerance of Pv11 cells. When a gene encoding a foreign protein is introduced into Pv11 cells linked to the vanderplanki 121 promoter, this promoter is constitutively activated, enabling effective expression of the foreign gene of interest.The high activity of the vanderplanki promoter allows for over 1500 times the protein production compared to promoters included in commercially available insect cell gene expression kits. To generate Pv11 cells stably expressing foreign genes, we used CRIS-PITCh knock-in to express GCaMP6f, Orco, Or47a, and Zeo. r The transgene was transfected into cells. Pv11 cell lines stably expressing the exogenous gene were established by selecting the transfected cells with Zeocin and cell sorting using GCaMP6f fluorescence. Functional expression of Orco and OR was confirmed by exposure to VUAA1, an Orco agonist, and odorants that bind to ORs (e.g., pentyl acetate, a ligand for Or47a). It was confirmed that the cells maintained their biological function after rehydration, even after repeated dehydration and rehydration over several years. Stable expression of OR-encoding genes knocked into the genome of Pv11 cells, enabling high expression, resulted in the selection of cells exhibiting strong responsiveness to specific ligands.

Claims

1. A cell derived from the sleeping chironomid (Polypedilum vanderplanki) expressing at least one foreign membrane protein.

2. The cell of claim 1 , wherein the exogenous membrane protein is functional when the cell is dried and then rehydrated.

3. The cell of claim 1 or 2, wherein the exogenous membrane protein comprises an olfactory receptor protein (OR) and / or an olfactory receptor protein co-receptor (Orco).

4. The cell of claim 3 , wherein the exogenous membrane proteins are OR and Orco.

5. The cell of claim 1 or 2, wherein the gene for the exogenous membrane protein is operably linked to a 121 promoter.

6. The cell of claim 1 or 2, further expressing one or more additional foreign proteins.

7. The cell of claim 6 , wherein the additional exogenous protein comprises a fluorescent marker protein that detects changes in intracellular calcium concentration.

8. The cell of claim 1 or 2, wherein the cell is derived from a Pv11 cell.

9. An odor sensor comprising cells derived from the vanderplanki expressing OR and Orco.

10. The odor sensor according to claim 9 , wherein the cells further express a fluorescent marker protein that detects changes in intracellular calcium concentration.

11. The odor sensor according to claim 9 or 10, wherein the cells are immobilized on a substrate.

12. An odor detection method comprising a step of detecting an odorant or identifying an odor using cells derived from the vanderplanki expressing OR and Orco.

13. A method for detecting odors, comprising the step of detecting odorants or identifying odors using a collection of cells derived from the vanderplanki expressing one or more ORs and Orco.

14. 1. A method for preserving an exogenous membrane protein, comprising: introducing a gene encoding the foreign membrane protein into a cell derived from Polypedilum vanderplanki; Culturing the cells to express the exogenous membrane protein in the cell membrane; drying the cells; A method comprising:

15. 15. The method of claim 14, further comprising suspending the cells in a solution containing a dry protectant prior to the drying step.

16. 1. A method for detecting or identifying an odor, comprising: (a) providing a desiccant-derived biosensor cell expressing one or more ORs and / or Orco; (b) contacting the biosensor cells with at least one test compound or sample; (c) detecting a signal indicative of activation of one or more ORs in the biosensor cell; A method comprising:

17. 1. A device for detecting the presence of an odorant or for identifying an odor, comprising: A device comprising desiccable insect-derived biosensor cells expressing one or more ORs that are activated upon contact with an odorant.

18. 18. The device of claim 17, which is a portable device.

19. A kit for the detection of odorants or for the identification of odors, comprising: A kit comprising one or more desiccable insect-derived biosensor cells.

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